Modified bacteriocins and methods for their use

ABSTRACT

Modified forms of naturally occurring bacteriocins, such as the R-type pyocins of  Pseudomonas aeruginosa , are disclosed as are methods for producing them in GRAS organisms. The bacteriocins are modified at the ends of their tail fibers in a region responsible for binding specificity and affinity to their cognate binding partners, or receptors, such as those on the surface of bacteria. Methods for the use of the modified bacteriocins, such as to bind receptors, including virulence or fitness factors, on the surfaces of bacteria, are also described.

RELATED APPLICATIONS

This application claims benefit to pending U.S. application Ser. No.11/748,432, filed May 14, 2007, which claims benefit to U.S. ProvisionalApplication 60/747,299, filed May 15, 2006, which are incorporated byreference as if fully set forth.

FIELD OF THE DISCLOSURE

This disclosure relates to modified forms of naturally occurring highmolecular weight (hmw) bacteriocins, such as the R-type pyocins ofPseudomonas aeruginosa. The bacteriocins are modified at the ends oftheir tail fibers in a region responsible for binding specificity andaffinity to their cognate binding partners, or receptors, such as thoseon the surface of bacteria. Methods for the use of the modifiedbacteriocins, such as to bind receptors, including virulence or fitnessfactors, on the surfaces of bacteria, are also described. Thisdisclosure also relates to R-type pyocins wherein the tail fibers aremodified to include globular proteins, which proteins can bind anddegrade cell surface structures, such as polysaccharides. Unnaturalsystems for production of R-type pyocins by bacterial cells generallyregarded as safe (“GRAS”) by regulatory authorities are described as areR-type pyocins produced by such GRAS bacteria.

BACKGROUND OF THE DISCLOSURE

Currently far more global attention is focused on threats from viralpathogens than from bacterial diseases. However, omnipresentantibiotic-resistant bacteria continue to wreak havoc on patient careand cost containment in hospitals and other medical care facilities. Atthe same time, there is a retreat from antibiotic development in favorof drugs for chronic diseases and life style improvements. In the lasttwenty years only two new classes of antibiotics (oxazolidinones andlipopeptides) have been introduced into the U.S. market (Wenzel, 2004).

In the United States alone, there are over 2 million cases of hospitalacquired bacterial infections every year. Of these, approximately 90,000people will die. The most alarming statistic is that over 70% of thesebacterial culprits are resistant to at least one antibacterial drug (BadBugs, No Drugs, 2004). This number continues to increase at an alarmingrate. The annual cost to the U.S. economy of these antibiotic-resistantnosocomial infections exceeds $5 billion. The reality of thisthreatening global situation will force a new approach to thedevelopment and use of antibacterial agents (Talbot et al., 2006). Whereextensive use (and abuse) of antibiotics in human and animal medicineflourished, so has the emergence of antibiotic-resistant bacterialpathogens to the point that many antibiotics that were once “wonderdrugs” are now clinically ineffective (Microbial Threats to Health,2003).

As one example, Pseudomonas aeruginosa is a ubiquitous pathogen forplants and animals that is exhibiting a rapidly rising incidence ofresistance to multiple antibiotic drugs (Microbial Threats to Health,2003; Bad Bugs, No Drugs, 2004). P. aeruginosa is an aerobic, motile,gram-negative, rod. P. aeruginosa normally inhabits soil, water, andvegetation. Although it seldom causes disease in healthy people, it isan opportunistic pathogen which accounts for about 10% of all nosocomialinfections (National Nosocomial Infection Survey report-Data Summaryfrom October 1986-April 1996). P. aeruginosa is the most common pathogenaffecting Cystic Fibrosis (CF) patients with 61% of the specimensculturing positive (Govan, J. R. W. and V. Deretic, 1996, Microbiol.Reviews, 60(3):530-574) as well as one of the two most common pathogensobserved in intensive care units (Jarvis, W. R. et al., 1992, J.Antimicrob. Chemother., 29(a supp.): 19-24).

Mortality from some P. aeruginosa infections can be as high as 50%.Presently, P. aeruginosa infection can still be effectively controlledby antibiotics, particularly by using a combination of drugs. However,resistance to several of the common antibiotics has been shown and isparticularly problematic in intensive care units (Archibald, L. et al.,1997, Clin. Infectious Dis., 24(2):211-215; Fish, D. N., et al., 1995,Pharmacotherapy, 15(3):279-291). Additionally, P. aeruginosa has alreadydemonstrated mechanisms for acquiring plasmids containing multipleantibiotic resistance genes (Jakoby, G. A. (1986), The bacteria, Vol. X,The biology of Pseudomonas, pp. 265-294, J. R. Sokach (ed.) AcademicPress, London) and at present there are no approved vaccines forPseudomonas infection.

Like many other bacterial species, strain variability in P. aeruginosais quite significant. Variability has been shown to occur by a number ofdifferent mechanisms, these include, but are not limited to, theintegration of prophages into a bacterial genome (Zierdt, C. H. and P.J. Schmidt, 1964, J. Bacteriol. 87:1003-1010), the addition of thecytotoxin gene from bacteriophages (Hayashi, T., et al., 1994, FEMSMicrobiol. Lett. 122:239-244) and via transposons (Sinclair, M. I. andB. W. Holloway, 1982, J. Bacteriol. 151:569-579). Through this type ofdiversity, new pathogenic mechanisms have been incorporated into P.aeruginosa. These and other transitions such as the conversion to themucoid phenotype, commonly seen in CF, clearly illustrate the need forcontinued vigilance.

These concerns point to the need for diagnostic tools and therapeuticsaimed at proper identification of drug-resistant strains and eradicationof virulence.

Many bacteria produce bacteriocins, which are bactericidal substances.Bacteriocins are composed of polypeptides and vary in molecular weight.While bacteriocins have been used for their antibacterial properties,some have more limited bactericidal spectra than many clinically usedantibiotics. For example some bacteriocins have been reported asrecognizing, and so acting on members of the same or closely relatedspecies by binding receptor sites on sensitive, or susceptible,organisms.

As a broad classification, bacteriocins have been divided into threetypes. The first are small molecules which are thermal stable. Examplesof this first type include Colicin V (where colicins are specific tocoliform bacteria). The second type, S-type pyocins produced by P.aeruginosa, are higher molecular weight protein molecules. The thirdtype includes bacteriocins that genetically and morphologically resemblethe tail portions of bacteriophages. Examples of this latter typeinclude the F-type and the R-type pyocins of P. aeruginosa as well asenterocoliticin of Yersinia. These pyocins have been reported as beingderived from ancestral bacteriophages. The F-pyocins have structuralsimilarities to the lambda phage family, and the latter two R-typepyocins are related to the P2 phage family.

R-type pyocins are similar to the non-flexible and contractile tailportions of bacteriophages of the myoviridae family and are encoded in asingle cluster of genes in the Pseudomonas genome (Shinomiya et al.,1983). See FIG. 1. After binding specifically to a target bacteriumthese pyocins form a pore in the bacterial cell, compromising theintegrity of its cytoplasmic membrane and causing membranedepolarization. F-type pyocins are also similar to a bacteriophage tail,but they have a flexible and non-contractile rod-like structure. Pyocinsare produced by the majority of P. aeruginosa strains, and some strainssynthesize more than one pyocin.

R-type pyocins are complex high molecular weight bacteriocins producedby some Pseudomonas aeruginosa strains, and have bactericidal activityagainst certain other P. aeruginosa strains (for a review seeMichel-Briand and Baysse, 2002). Five R-type pyocins have beenidentified to date and, based on their target spectra (see below), aretermed R1 through R5. Strain PAO1 produces R2 pyocin, which is encodedin a gene cluster consisting of 16 open reading frames (ORFs), 12 ofwhich show significant sequence similarity to ORFs of bacteriophages P2,PS17, ΦCTX, and other P2-like phages (Nakayama et al., 2000). Pyocinproduction is induced by DNA damage (Matsui et al., 1993) and isregulated by RecA, which degrades PrtR, the repressor of PrtN, apositive transcription regulator of the cluster. Induction of pyocingenes results in synthesis of approximately 200 pyocin particles perbacterial cell followed by lysis of the cell by mechanisms similar tothose of bacteriophage lysis. Pyocins rapidly and specifically killtarget cells by first binding to the lipopolysaccharide (LPS) via theirtail fibers, followed by sheath contraction and core penetration throughthe bacterial outer membrane, cell wall and cytoplasmic membrane. Thispenetration compromises the integrity of the cytoplasmic membrane anddepolarization of the membrane potential (Uratani and Hoshino, 1984). Inmany respects pyocins can be viewed as defective prophages adapted bythe host to produce protease- and acid-resistant, noninfectiousantibacterial particles consisting only of the adapted tail apparatus,that is, without capsids or DNA. The replication of the pyocin genesrequires the replication of the bacterial genome in which they areembedded.

The five different pyocin receptor specificities are related linearly toone another with two branches. (Ito et al, 1970; Meadow and Wells, 1978;Kageyama, 1975). R5 pyocin has the broadest spectrum and includes thespecificities of the other four. The receptors for the other fourR-types form two branches, or families of specificities, that divergefrom R5. One branch includes the receptors for R3, R4, and R2, in thatorder where the receptor specificity for R3 pyocin is the most distalfrom the cell surface. The second branch contains the R1 receptor, whichseems to have a specificity determinant unrelated to those for R2, R3,and R4. The two branches seem to be attached to the receptor for R5since all P. aeruginosa strains that are sensitive to any of R1-R4pyocins are sensitive also to R5, while some strains are sensitive onlyto R5 pyocin. Some P. aeruginosa strains are resistant to all 5naturally occurring R-type pyocins.

P. aeruginosa pyocins specifically kill mainly strains of P. aeruginosabut have also been shown to kill some strains of Hemophilus, Neisseriaand Campylobacter species (Filiatrault et al., 2001; Morse et al, 1976;Morse et al, 1980; Blackwell et al., 1981, 1982).

The specificity of R-type pyocins is conferred by the tail fiber encodedby the gene: prf15. PRF15 protein is very closely related to the tailfibers of phages of the Myoviridae family, particularly P2-like phages(Nakayama et al., 2000). These tail fibers are homotrimers arrangedsymmetrically on a base plate structure with six copies per particle, asshown in FIG. 1. The N-terminal region of the tail fiber binds to thebaseplate, and the C-terminal portion, probably near the tip, binds tothe bacterial receptor and thereby confers killing specificity. Acognate chaperone, PRF16 protein, encoded by prf16 gene (in the case ofR-type pyocins) is located immediately downstream of prf15, and isneeded for proper folding of the tail fiber and/or assembly of the tailfibers on the pyocin structure. R-type pyocin particles have beendescribed as immunochemically and genetically similar to the tails ofcertain P. aeruginosa bacteriophages (Kageyama 1975, Kageyama et al.1979, Shinomiya et al. 1989, and Shinomiya et al. 1983b). It has beenproposed that R-type pyocins and Pseudomonas bacteriophages, such asPS-17 and ΦCTX, are related through a common ancestral lysogenicbacteriophage from which genes encoding head proteins and replicationfunctions were lost and the residual phage genes adapted for theirfunction as components of the defensive R-type pyocins (Shinomiya et al.1989).

Similar R-type high molecular weight bacteriocins have been described inother bacteria including Yersinia enterocolitica (Strauch et al., 2001),Listeria monocytogenes (Zink et al, 1995), Staphylococcus aureus(Birmingham & Pattee, 1981) and Erwinia amylovora (Jabrane et al.,2002). Classification and nomenclature of bacteriocins have undergonechanges over time, particularly given expanding evidence of theirorigin, chemistry and activities. Typically, the naming of bacteriocinsis based on the producing species. For example, E. coli producesbacteriocins termed colicins; Pseudomonas aeruginosa produces pyocins;Listeria monocytogenes produces monocins; Yersinia enterociliticusproduces enterocoliticins; and so forth. Historically, theclassification began with the identification of about 20 colicins whichwere classified as A-V. In most cases, each bacteriocin appears to bespecific in action to the same, or to taxonomically related, species oforganisms. Pyocin-producing strains typically are resistant to their ownpyocin. A general assay for the concentration of bacteriocin isdescribed in U.S. Pat. No. 4,142,939.

Certain pathogenic E. coli strains, such as E. coli O157:H7, arefood-borne pathogens. Outbreaks of illnesses from E. coliO157:H7-contaminated meats, raw vegetables, dairy products, juices, andthe like, have caused considerable morbidity and mortality. Agents andmethods are needed to effectively and safely sterilize or sanitize foodproducts that could be contaminated with these pathogenic bacteria.

Citation of the above documents is not intended as an admission that anyof the foregoing is pertinent prior art. All statements as to the dateor representation as to the contents of these documents is based on theinformation available to the applicant and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

SUMMARY OF THE DISCLOSURE

This disclosure relates to engineered forms of the class of bacteriocinsthat resemble, but are distinct from, bacteriophage tails. Thesebacteriocins include R-type pyocins, tail-like bacteriocins, R-typebacteriocins, or other high molecular weight (hmw) bacteriocins relatedto the tail structures of bacteriophages. For ease of reference, theterm “hmw bacteriocin” will be used herein to refer to the bacteriocinsof the disclosure, including, but not limited to, R-type bacteriocins,R-type pyocins, monocins, enterocoliticins, meningocins and competencefactor of Staphylococcus aureus (Thompson and Pattee, 1981).

Natural hmw bacteriocins are typically thermolabile, trypsin resistant,and can be induced by agents, which activate the SOS system. Forexample, they also have been identified in many enterobacteria,Pseudomonas species, Rhizobium lupin, Listeria monocytogenes, Bacillusspecies, Yersinia species, Erwinia species, and Flavobacterium species.

An engineered hmw bacteriocin is composed of multiple copies of a numberof different polypeptide subunits and possesses one or more, usuallysix, tail fibers made up of tail fiber proteins. Each tail fibercontains a receptor binding domain (RBD) which binds to, or interactswith, a receptor to form a binding pair. The RBD is the portion of atail fiber that comprises the bacteria binding property that makes itthe first member of the binding pair. An RBD as disclosed hereincomprises modification of a protein in the tail fiber to form a modifiedtail fiber. The modified tail fiber with the other polypeptide subunitsforms an engineered (or modified) hmw bacteriocin. The receptor to whichthe RBD binds is the second member of the binding pair, and may be thesame as, or different from, the receptor for a bacteriocin without themodified tail fiber. In some embodiments of the disclosure, the secondmember of a binding pair is a virulence or fitness factor of apathogenic bacterium. In other embodiments, the second member is acomponent of the outermost layer(s) of a bacterial cell, such as a cellmembrane or, in the case of gram-positive bacteria, cell wall component.

In comparison to an hmw bacteriocin lacking the modified tail fiber, anengineered hmw bacteriocin may differ in the number, manner, and bindingstrength of its interactions with a receptor. Thus an engineered hmwbacteriocin may have different or additional binding properties (e.g.binding specificities, affinities, and/or avidities) in comparison to abacteriocin without the modification. An engineered hmw bacteriocin isnot a naturally occurring molecule but may be a modified version of anaturally occurring molecule. Alternatively, an engineered hmwbacteriocin may be a modified version of another non-naturally occurringbacteriocin. In most embodiments, an engineered hmw bacteriocin remainsa lethal agent for bacterial cells expressing a receptor bound by thebacteriocin.

In a first aspect, the disclosure includes an hmw bacteriocin comprisinga tail fiber protein with a modified RBD. Non-limiting examples of hmwbacteriocins include R-type pyocins. In some embodiments, the modifiedRBD comprises a change in the amino acid sequence of the domain relativeto a naturally occurring bacteriocin. Non-limiting examples of a changein amino acid sequence include substitution, insertion (addition), ordeletion of one or more amino acids. Of course combinations of one ormore substitutions, insertions (additions), and deletions may also beused.

In other embodiments, the tail fiber comprises a heterologous, ornon-bacteriocin, sequence in one or more of the three tail fiber proteinmonomers that make up a single trimeric tail fiber. And while the tailfibers in a native, or naturally occurring, bacteriocin may behomotrimeric to form an RBD, the tail fiber of an engineered hmwbacteriocin is either heterotrimeric, where one or two of the proteinmonomers is different from the other(s), or homotrimeric where all threeprotein monomers are identically non-native (non-naturally occurring).The presence of heterologous (or non-native) sequence, in one or moreprotein monomers allows the trimer to form a tail fiber with a modifiedRBD.

The heterologous sequence is thus in a part of the monomer(s) such thatat least the RBD of the tail fiber is altered in an assembled trimer.The altered RBD changes the binding characteristics and properties ofthe tail fiber and thereby the binding activity of a hmw bacteriocincontaining the tail fiber. In some embodiments, the heterologous RBD isderived from another bacteriocin or a tail protein from a bacteriophageor prophage. In many cases, the heterologous RBD is a polypeptideincluding at least part of the C-terminal portion of a tail fiberprotein of a bacteriocin, a bacteriophage tail fiber protein, or apresumptive tail fiber protein, the sequence of which has been derivedfrom a gene of a viable or even defective lysogenic bacteriophage foundwithin the genome of a bacterium.

The heterologous RBD can be derived from a bacteriophage that encodes atail protein or a tail spike-like protein, which protein is globular andpreferably, polysaccharide specific. Tail spikes are tail components,usually homotrimeric in structure, such as those found in bacteriophagesP22 and epsilon 15. They perform similarly to tail fiber proteins andcan be engineered. Therefore, for the purposes of this disclosure, tailspikes or portions thereof are considered RBDs. These proteins can bindand degrade cell surface polysaccharide structures. The same motif ofthe tail spike protein structure that binds the enzyme substrate canprovide the RBD function, since the substrate for the enzyme may alsoserve as the receptor on the surface of the target bacteria. Forexample, phiV10 is a bacteriophage belonging to the podoviridae group,and can infect E. coli O157:H7 strains. (Waddell & Poppe 2000; GenbankNC_(—)007804). The phiV10 tail spike (SEQ ID NO: 60) specificallyrecognizes, binds to, and degrades the 0157 antigen on the surface ofpathogenic E. coli O157:H7. Other globular proteins with polymer bindingand degrading activities, such as but not limited to, SEQ. ID NO.: 61,62, 63, 64, and 65 can serve as RBD structures in engineered hmwbacteriocins.

The tail spike of the CUS3 prophage of K1 strain of E. coli RS218, SEQ.ID. NO.:61 is an endosialidase very similar to that of the tail proteinsof phages K1E, and K1F that recognize and degrade the capsule of K1strains of E. coli. The CUS3 tail spike has a head binding domainsimilar to the P22 tail spike protein and a C-terminus related to theother phage endosialidases. Fusing this tail spike or a portion thereofto the R2 tail fiber base plate attachment region (BPAR) will create apyocin that can kill K1 strains of E. coli.

The tail spike of bacteriophage HK620, SEQ. ID. NO.:62, is the tailspike of HK620, a P22-like phage. It is specific for the O-antigen of E.coli H. Fusing this tail spike or a portion thereof to the R2 tail fiberBPAR will create a pyocin that can kill H strains of E. coli.

The tail spike of phage Sf6, SEQ. ID. NO.:63, is also P22-like andrecognizes and degrades the Shigella flexneri O-antigen. Fusing thistail spike or a portion thereof to the R2 tail fiber BPAR will create apyocin that can kill Shigella flexneri strains.

The tail spike of phage ST64T, SEQ. ID. NO.:64, is P22-like, andrecognizes the O-antigen of Salmonella typhimurium DT64. Fusing thistail spike or a portion thereof to the R2 tail fiber BPAR will create apyocin that can kill Salmonella typhimurium strains such as DT64.

Phage epsilon15 tail spike, SEQ. ID. NO.:65, is similar to the phiV10tail spike and specific for group E1 Salmonella enterica. Fusing thistail spike or a portion thereof to the R2 tail fiber BPAR will create apyocin that can kill group E1 Salmonella enterica strains.

Tail spike proteins and portions thereof may fold properly without thefacilitation by a specific chaperone. Thus, the generation of fusionsbetween an R-type pyocin tail fiber and a heterologous tail spikeprotein as an RBD may not require an RBD-specific chaperone as usuallydoes the fusion between an R-type pyocin tail fiber and a heterologoustail fiber protein from another R-type pyocin or a myoviridae such asP2, L-413c, AB17 or PS17.

The heterologous RBD is fused to a polypeptide containing a BPAR of ahmw bacteriocin or a myoviridae phage tail fiber protein. The BPARcontaining polypeptide may contain all or part of the N-terminal portionof an hmw bacteriocin tail fiber, where the N-terminal portion canconsist of any part of the tail fiber except the very C-terminus.

In other embodiments, the heterologous RBD is derived from the majortropism determinant (MTD) of Bordetella bacteriophage. Non-limitingexamples include a heterologous RBD comprising a modified or diversifiedtropism determinant, optionally with all or part of the RBD of a tailfiber of a bacteriophage. In some embodiments, the bacteriophage tailfiber is that of the Vibrio harveyi myovirus-like (VHML) bacteriophageor its diversified derivatives or those of another prophage orbacteriophage that comprises a Diversity Generating Retroelement (DGR)structure.

The disclosure further includes a portion of an engineered hmwbacteriocin where the portion retains the bacteriocin's activity ofbinding a receptor on a bacterial cell surface and then promoting thepenetration of the cell membrane. Thus the portion may be any thatretains the binding (recognition) and membrane penetration activities ofan engineered hmw bacteriocin. In some embodiments, the portioncomprises one or more bacteriocin polypeptides that are truncated.

In a related aspect, the disclosure includes modified tail fibers thatmay be part of an hmw bacteriocin of the disclosure. The trimeric tailfiber may comprise one or more tail fiber proteins with a modified RBDor a heterologous RBD. In some embodiments, the modified monomeric tailfiber protein is derived from an R-type bacteriocin while in otherembodiments, the tail fiber protein is derived from a bacteriophage tailfiber or a bacteriophage tail spike protein.

The disclosure also includes native, isolated, or recombinant nucleicacid sequences encoding a modified tail fiber protein, as well asvectors and/or (host) cells containing the coding sequences. The vectorsand/or host cells may be used to express the coding sequences to producenative, isolated, or modified tail fiber proteins which form tail fibersand are incorporated into an engineered hmw bacteriocin of thedisclosure. A sequence encoding a modified tail fiber protein may alsobe introduced into a bacterial cell which produces, or is capable ofproducing, an hmw bacteriocin in the presence of the modified tail fiberprotein.

In some instances the bacterium for production will be those designatedas “generally recognized as safe,” or “GRAS,” under the U.S. FederalFood, Drug, and Cosmetic Act, such as for food additives or foodingredients. Typically any substance that is intentionally added to foodis a food additive that is subject to review and approval by the U.S.Food and Drug Administration (FDA) unless the substance is generallyrecognized by experts as having been adequately shown to be safe underthe conditions of its intended use. A GRAS substance can be utilizedwithout pre-approval.

Expression of the modified tail fiber protein results in the productionof a modified hmw bacteriocin by the cell. If natural bacteriocin tailfiber protein sequence(s) or the nucleic acid(s) encoding such proteinis/are inactivated or removed, then only modified hmw bacteriocins willbe produced. If natural bacteriocin tail fiber protein sequence(s) orthe nucleic acid(s) encoding such protein are retained, then modifiedhmw bacteriocins will be produced along with the natural bacteriocintail fibers, and the modified pyocins generated may be mixtures of bothmodified pyocins and natural pyocins. In addition, the pyocins generatedfrom such production host bacteria may contain bivalent (multivalent)pyocins, that is, contain single pyocin particles with a mixture of twotypes of tail fibers, each with its specific binding properties. Suchmultivalent pyocins have multiple, that is, two or more, binding andkilling specificities within the same pyocin particle or molecule. Thetransfected bacteria may be propagated to produce hmw bacteriocins thatprevent or inhibit the growth of other bacteria that express a receptorbound by the modified hmw bacteriocin or by one of the hmw bacteriocinsfrom the mixture of natural plus modified hmw bacteriocins.

In some embodiments, the receptor is a virulence or fitness factor of avirulent or pathogenic bacterial strain such that exposure to themodified hmw bacteriocin prevents or inhibits growth of the virulent orpathogenic strain. Non-limiting examples of virulence factors targetedby an engineered hmw bacteriocin include those encoded by the sequencesdisclosed in U.S. Pat. No. 6,355,411 and published patent application WO99/27129 (Ausubel et al.).

The exposure is optionally via contact, or co-culturing, withtransfected bacteria expressing the hmw bacteriocin. The disclosureincludes allowing propagation of the transfected bacteria in vivo, on orwithin an animal or plant subject. The in vivo application of thetransfected bacteria provides a state of protection against bacteriaexpressing a surface receptor targeted by the engineered hmwbacteriocin. The state of protection is analogous to a state ofimmunity, where the transfected bacteria essentially augment orsupplement the animal or plant organism's immune or other defensesystem.

In other embodiments, the nucleic acid sequence coding an RBD of amodified monomeric tail fiber protein is part of a genetic system whichpermits the identification, physical isolation and/or selection of thecoding sequence. As non-limiting examples, the genetic system maycomprise the coding sequence in a phage, lysogenic phage, transducingparticle, cosmid, or phage genome allowing its identification,isolation, and/or selection. In some embodiments, the sequence is fusedwith a portion of a fiber gene and expressed to produce a modified tailfiber trimer that will cause the modified hmw bacteriocin to bind to thesurface of and kill the host organism harboring the lysogenic phage fromwhich the RBD coding sequence was identified or isolated. Detection of aphenotype in the modified tail fiber trimer permits the sequence to beselected and/or screened, identified, and isolated. In some embodiments,the phenotype may be a desired, and possibly rare, receptor-bindingproperty.

The disclosure includes a library of phages, transducing particles,cosmids, or phage genomes, containing a plurality of DNA and/or RNAsequences, each encoding a modified tail fiber protein. This coupling ofbinding phenotype to encoding genotype of the RBD allows the expressionof a plurality of modified RBDs such that the sequences encoding themare represented within the library. In some embodiments, the members ofa library each contain a sequence encoding one modified tail fiberprotein such that homotrimeric tail fibers are expressed and availablefor screening or selection to determine the respective binding phenotypeof a library member. In other embodiments, the members of a libraryinclude those with more than one sequence encoding a modified tail fiberprotein such that heterotrimeric tail fibers disclosed herein may beexpressed and screened or selected for their binding phenotypes. Thebinding phenotype of a member of the library is thus coupled to therespective coding sequence(s). Once the genotype encoding the desired oradvantageous RBD has been so identified, it can be used to create thetail fiber for a modified hmw bacteriocin. By deploying the cognatechaperone function of a tail fiber, such as that of VHML, that naturallydiversifies its RBD, one can be assured of proper folding of a tailfiber containing a diversified RBD derived from VHML.

Vectors, host cells, phages, transducing particles, cosmids, phagegenomes, and libraries as disclosed herein may be consideredcompositions comprising a tail fiber protein encoding nucleic acidmolecule.

Compositions of the disclosure also comprise fusion proteins resultingfrom the fusion of the RBD protein to the BPAR protein. For example, allor part of the phiV10 tail spike is fused to the BPAR of the R-typepyocin tail fiber PRF15. In some instances, these fusion proteins can beprovided in the context of other proteins, or phage or cellularcomponents. Alternatively, they may be isolated or separated. The fusionproteins can be part of a library and available for screening orselection, and/or may be associated with a carrier or excipient foradministration. They can be prepared via recombinant methods orsynthesized chemically.

Additional compositions of the disclosure comprise an engineered hmwbacteriocin or an anti-bacterial portion thereof. The compositions areanti-bacterial by virtue of the hmw bacteriocin, and may comprise acarrier or excipient. Of course the carrier or excipient is one that issuitable for use in combination with a multisubunit complex protein likean hmw bacteriocin. In some embodiments, the carrier or excipient ispharmaceutically acceptable such that the composition may be usedclinically or agriculturally. In other embodiments, the carrier orexcipient is suitable for topical, pulmonary, gastrointestinal, orsystemic administration, such as to a human or a non-human animal. Inadditional embodiments, the carrier or excipient is suitable foradministration to a surface or to a non-animal organism such as a plantor fresh produce from a plant as non-limiting examples.

A composition as disclosed herein may comprise more than one fusionprotein or engineered hmw bacteriocin or comprise one or more additionalagents, including but not limited to, a naturally occurring hmwbacteriocin desired for use with the engineered hmw bacteriocin.Non-limiting examples of an additional agent include an enzyme, anantibiotic, an anti-fungal agent, a bactericide, an analgesic, and ananti-inflammatory agent.

In a further aspect, the disclosure provides methods of using an hmwbacteriocin related product described herein. Embodiments of thedisclosure include methods of inhibiting bacterial cell growth orinducing bacterial cell death. Such methods comprise contacting asusceptible bacterial cell or cells with an effective amount of anengineered hmw bacteriocin, or with an anti-bacterial portion thereof,such as a fusion protein. Alternatively a composition containing the hmwbacteriocin, or anti-bacterial portion thereof, may be used. In somecases, an effective amount may be equivalent to as few as one, onaverage, hmw bacteriocin per bacterial cell. Higher amounts also may beused.

In other embodiments, a method of compromising the integrity of thecytoplasmic membrane of a bacterium is provided. The compromise mayresult in the loss of membrane potential and/or loss of some cellularcontents. Such methods comprise contacting the membrane with a fusionprotein, or an engineered hmw bacteriocin, or anti-bacterial portionthereof. In many cases, the membrane will be that of virulent orpathogenic bacteria.

In some embodiments, the methods of the disclosure may comprise in vivoapplication (or administration) of a fusion protein or an engineered hmwbacteriocin, or an anti-bacterial portion thereof, within a subject.Alternatively, the methods may comprise in vitro or ex vivo contacting.

In a yet additional aspect, the disclosure provides a method of formingnon-virulent bacteria from virulent progenitor bacteria. The methodcomprises contacting virulent bacteria with an engineered hmwbacteriocin, or an anti-bacterial portion thereof, which binds avirulence or fitness factor of the virulent bacteria. The contacting maybe under conditions wherein not all of the bacteria are killed, orwholly inhibited in cell growth, by the used amount of hmw bacteriocin,or anti-bacterial portion thereof. The contacting provides a selectivepressure that allows the targeted bacterium to survive the engineeredhmw bacteriocin or anti-bacterial portion thereof and to propagate onlyif it has become a non-virulent mutant or modified bacteria progeny thatis not susceptible (and so resistant) to the engineered hmw bacteriocinor anti-bacterial portion thereof. In some embodiments, the resistanceis due to the lack of expression of the virulence or fitness factor orreceptor for the engineered hmw bacteriocin, or anti-bacterial portionthereof, thereby avoiding attack by the engineered hmw bacteriocin. Inanother embodiment the resistance may be due to an alteration in thevirulence or fitness factor such that it no longer serves as aneffective receptor for the RBD of the modified pyocin and in the alteredform also compromises its virulence or fitness function. The acquisitionof resistance by the surviving progeny, and the resultant change invirulence or fitness of a formerly virulent bacterium, can be determinedin vivo or in vitro to demonstrate its compromised pathogenicity.

In a related aspect, the disclosure provides a method of maintaining apopulation of non-virulent bacteria by contact with an engineered hmwbacteriocin, or an anti-bacterial portion thereof, which binds to andmediates its bactericidal effect via a virulence or fitness factor ofthe virulent bacteria. The presence of the hmw bacteriocin preventsgrowth (or generation or propagation) of virulent bacteria and somaintains the population as non-virulent. In some embodiments, thecontacting may be by use of a bacterial cell, as described herein, whichexpresses the engineered hmw bacteriocin or anti-bacterial portionthereof.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages of the disclosure will be apparent from thedrawings and detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the electron micrograph of an R-type pyocin particlerevealing 4 of the 6 tail fibers in Panel A, and a schematic of themajor components of an R-type pyocin particle in Panel B.

FIG. 2 provides spot serial (5×) dilution assays of wild type pyocins(R2), pyocin particles produced from the tail fiber deletion strain(PA01Δprf15), and pyocins complemented with the R2-P2 tail fiber fusion.Target bacteria are P. aeruginosa 13s and E. coli C1a. Wild type R2pyocin particles can kill Pseudomonas but not E. coli. The tail fiberdeletion strain produces no active pyocin particles, but whencomplemented in trans with the R2-P2 tail fiber fusion, it now can killE. coli C1a.

FIG. 3 is complementing the R2 pyocin structure with an R2-P2 tail fiberfusion. The C-terminal (RBD) portion of the P2 tail fiber gene was fusedto the N-terminal (BPAR) portion of the R2 tail fiber, as shown in partA.

Part B of FIG. 3 shows a schematic of the wild type R2 pyocin (left).The R2 pyocin is complemented with the R2 (BPAR)—P2 (RBD) fusionconstruct to produce particles (right) that have the chimeric tailfibers incorporated into the structure. The R2-P2 particles have analtered killing spectrum and now target certain E. coli strains.

FIG. 4 provides a multiple R2-P2 fusions and their bactericidalactivities. The N-terminus, 1-164 amino acids, of R2 (Base-Plate BindingRegion, “BPAR”) was fused to various C-terminal portions of P2 (RBD).The numbers represent the amino acid reside numbers of the respectiveproteins. The bactericidal activity of the modified pyocins (against E.coli C) containing each of the constructed tail fibers are indicated aspresent (+) or absent (−).

FIG. 5 shows various portions of the N-terminus of the R2 tail fiber(BPAR) fused to the C-terminal 158-669 portion (RBD) of the P2 tailfiber. The numbers represent the amino acid reside numbers of therespective proteins. The bactericidal activity of the modified pyocins(against E. coli C) containing each of the constructed tail fibers areindicated as present (+) or absent (−).

FIG. 6 shows multiple R2-P2 fusions and their bactericidal activities.N-terminus, 1-240 amino acids, of R2 (BPAR) was fused to variousC-terminal portions of P2 (RBD). The numbers represent the amino acidreside numbers of the respective proteins. The bactericidal activity ofthe modified pyocins (against E. coli C) containing each of theconstructed tail fibers are indicated as present (+) or absent (−).

FIG. 7 provides various portions of the N-terminus of the R2 tail fiber(BPAR) fused to the C-terminal 322-669 portion (RBD) of the P2 tailfiber. The numbers represent the amino acid reside numbers of therespective proteins. The bactericidal activity of the modified pyocins(against E. coli C) containing each of the constructed tail fibers areindicated as present (+) or absent (−).

FIG. 8 shows the trans complementation of the PA01Δprf15 R2 pyocinstructure with various R-type pyocin tail fibers, tail fiber fusions andchaperones. Activities of the R1 through R5 complemented pyocins wereassessed by spotting onto indicator strain Pseudomonas aeruginosa 13s,which is sensitive to all pyocin types. The R2-P2 complemented pyocinswere tested for activity using E. coli C as the indicator, and theR2-L-413c complemented pyocin was tested on Yersinia pestis strain KIM.

The R2, R3, and R4 PRF15 tail fibers could be complemented by theendogenous PRF 16 of the PA01Δprf15 R2 pyocin. R1 and R5 PRF 15 tailfibers, which differ at the C-terminus compared to R2, required, formaximal activity, their own cognate PRF 16 (which itself differs fromthe R2 counterpart). Both the R2-P2 and R2-L-413c fusions, which containthe C-terminus (RBD) of the phage P2 and L-413c tail fibers,respectively, require their cognate tail fiber assembly chaperonesencoded by gene G of the phage.

FIG. 9 shows the pyocin tail fiber and chaperone expression vectorpUCP30T. The genes, prf15 and prf16, are expressed using aPseudomonas/E. coli shuttle vector (Schweitzer) with replication origins(ori pRO1600, rep, and oriT) for both species. Cloning sites are shownby the indicated restriction enzyme sites of cleavage. The plasmidconfers gentamicin resistance (Gm R) and is maintained by addinggentamicin to the culture media. Transcription of both genes is drivenby the tac promoter which is negatively regulated by lacI^(Q). Whentransformed into Pseudomonas aeruginosa strain PA01Δprf15, the genes,e.g. prf15 and prf16, incorporated into the plasmid are expressed intrans after being induced with IPTG simultaneously with the mitomycin Cinduction of those pyocin genes remaining in the PAO1Δprf15 hostproduction bacteria.

FIG. 10 provides the construction of Yersinia pestis specific pyocintail fiber. Similar to the strategy that was used to construct R2-P2,the C-terminal (RBD) encoding portion of the L-413c tail fiber gene wasfused to an N-terminal portion (BPAR) of the R2 tail fiber. Whenexpressed in trans to complement the R2 tail fiber deletion strainPA01Δprf15, modified pyocin particles are produced containing thechimeric R2-L-413c tail fibers that can efficiently kill Y. pestis butnot Pseudomonas.

FIG. 11A shows representative tail fiber fusions of the bacteriophagesP2 and phiV10 tail fiber receptor binding domains (RBD) to the R2 pyocintail fiber base plate attachment region (BPAR).

FIG. 11B provides bactericidal activities of pyocins that haveincorporated either the R2-P2 tail fiber fusion or the R2-V10 tail fiberfusion into their structures. Pyocins were produced by expressing thetail fiber fusions in trans in PA01Δprf15 while simultaneously inducingthe pyocin genes. Pyocin activity was assessed by the spot method onlawns of E. coli EDL933 and TEA026. EDL933 is a wild type strain thatproduces the 0157 antigen. TEA026 is a mutant of EDL933 defective inO-antigen production (Ho and Waldor, 2007). EDL933 is sensitive toR2-V10 but not R2-P2. TEA026 is sensitive to pyocin R2-P2 but notR2-V10. This indicates that the 0157-antigen is the receptor for the V10RBD and that the P2 RBD recognizes the lipopolysaccharide.

FIG. 12 is a cartoon of the P4 plasmid, pDG218, containing the genes forgentamicin resistance (aacC1) and a fusion tail fiber such as P2H-V10inserted in the non-essential region of the P4 satellite phage anddriven by the left early promoter, P_(LE). The other indicated genes andfunctions are from P4sid₁ (Shore et al., 1977).

FIG. 13 provides the amino acid sequences or nucleic acid sequences forSEQ ID NOS:1-71, provided on pages 13A-13N.

DEFINITIONS

As used herein, an hmw bacteriocin includes an R-type pyocin, tail-likebacteriocin, R-type bacteriocin, and R-type pyocins, monocins,enterocoliticins, meningocins, or other high molecular weight (hmw)bacteriocins. An hmw bacteriocin includes modified versions of R-typepyoc ins, enterocoliticins, monoc ins, and meningoc ins (see Kingsbury“Bacteriocin production by strains of Neisseria meningitidis.” J.Bacteriol. 91(5):1696-9, 1966). A modified or engineered hmw bacteriocinmay be a modified R-type pyocin selected from the R1, R2, R3, R4, or R5pyocin of P. aeruginosa. The modified or engineered bacteriocins mayinclude the tail spikes of a bacteriophage, such as phiV10.

A bacteriocin of the disclosure may be thermolabile, mild acidresistant, trypsin resistant, sedimentable by centrifugation at about65,000×g, and resolvable by electron microscope (see Jabrane et al.Appl. Environ. Microbiol. 68:5704-5710, 2002; Daw et al. Micron27:467-479, 1996; Bradley Bacteriol. Revs. 31:230-314, 1967; andKageyama et al. Life Sciences 9:471-476, 1962. In many cases, anengineered hmw bacteriocin disclosed herein has one or more, in anycombination, of these properties. An additional property common tobacteriocins and engineered hmw bacteriocins disclosed herein is thatthey do not contain nucleic acid and thus are replication deficient suchthat they cannot reproduce themselves after or during the killing of atarget bacterium as can many bacteriophages.

R-type pyocins, and other hmw bacteriocins disclosed herein, are complexmolecules comprising multiple protein, or polypeptide, subunits andresemble the tail structures of bacteriophages of the myoviridae family.In naturally occurring R-type pyocins, the subunit structures areencoded by the bacterial genome such as that of P. aeruginosa and formpyocins to serve as natural defenses against other bacteria (Kageyama,1975). A sensitive, target bacterium can be killed by a single R-typepyocin molecule (Kageyama, 1964; Shinomiya & Shiga, 1979; Morse et al.,1980; Strauch et al., 2001).

A “target bacterium” or “target bacteria” refer to a bacterium orbacteria that are bound by an engineered hmw bacteriocin of thedisclosure and/or whose growth, survival, or replication is inhibitedthereby. The term “growth inhibition” or variations thereof refers tothe slowing or stopping of the rate of a bacteria cell's division orcessation of bacterial cell division, or to death of the bacteria.

As used herein, a “nucleic acid” typically refers to deoxyribonucleotideor ribonucleotides polymers (pure or mixed) in single- ordouble-stranded form. The term may encompass nucleic acids containingnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding, structural, or functional properties as the referencenucleic acid, and which are metabolized in a manner similar to thereference nucleotides. Non-limiting examples of such analogs include,without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-0-methyl ribonucleotides,and peptide-nucleic acids (PNAs). The term nucleic acid may, in somecontexts, be used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A particular nucleic acid sequence also encompasses conservativelymodified variants thereof (such as degenerate codon substitutions) andcomplementary sequences, as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third (“wobble”) position of one ormore selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues. Thus a nucleic acid sequence encoding a proteinsequence disclosed herein also encompasses modified variants thereof asdescribed herein.

The terms “polypeptide”, “peptide”, and “protein” are typically usedinterchangeably herein to refer to a polymer of amino acid residues.Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission.

Virulence factors are those molecules that contribute to thepathogenicity of an organism but not its general viability. Upon theloss of a virulence factor the organism is less pathogenic but notnecessarily less viable. Virulence factors may have any one of numerousfunctions, for example, regulating gene expression, providing adhesionor mobility, pumping out antibiotic agents, or forming protectivecoatings including biofilms.

Fitness factors are those molecules that contribute to the organism'sgeneral viability, growth rate or competitiveness in its environment.Upon the loss of a fitness factor, the organism is less viable orcompetitive and because of this compromise, indirectly less pathogenic.Fitness factors may also possess any one of numerous functions, forexample, acquiring nutrients, ions or water, forming components orprotectants of cell membranes or cell walls, replicating, repairing ormutagenizing nucleic acids, providing defense from or offense towardsenvironmental or competitive insults.

Some virulence and fitness factors are present on the surface of thebacterium and thereby accessible to an hmw bacteriocin disclosed herein.By binding to some surface virulence or fitness factors, an hmwbacteriocin can mediate killing by puncturing the cell membranes,compromising the integrity of the cytoplasmic membrane and/ordissipating the membrane potential of the cell. Those surface accessiblemolecules most likely to support hmw bacteriocin binding and killing areproteins, polysaccharides, and lipopolysaccharides of the outermembrane. Accordingly, potential targets for engineered hmw bacteriocinsare virulence factors and fitness factors that are proteins,polysaccharides and lipopolysaccharides of the outer membrane. Somenon-limiting examples of virulence factor targets for engineered pyocinsinclude intramembrane cleaving protease (iCLIP) metalloproteases; IL andIIL galactose- and fucose-binding lectins; microbial surface componentsrecognizing adhesive matrix molecule (MSCRAMM) proteins; and adhesin,such as ACE.

The ultimate success of targeting a specific virulence factor depends onits topography on the bacterial surface, its density on the surface,perhaps its two-dimensional mobility within the outer membrane, and itsprevalence in clinical or field isolates of the pathogen. For example,OprM is a porin-like outer membrane protein involved in multiple effluxpumps, e.g. the MexAB system, and prevalent in many gram-negativebacteria (Wong and Hancock, 2000). TolC, similar to OprM, is a requiredaccessory protein for many efflux pumps of gram-negative pathogens(Koronakis et al., 2004; Piddock, 2006). In addition, several members ofthe YcrC family of secretins are outer membrane proteins necessary forthe translocation of pathogenic effector proteins by the type threesecretion system (“T3 SS”), on which many gram-negative pathogens suchas P. aeruginosa and Yersinia pestis are dependent for intoxicatingtheir mammalian host (Galan and Collmer. 1999; Koster et al., 1997;Cornelis, 2006). In addition, the YscW family members are lipoproteinsalso anchored in the outer membrane to assist the insertion of thesecretins into the membrane (Burghout et al., 2004).

Additional non-limiting examples of virulence and fitness factorsinclude an aquaporin, such as the E. coli aquaporin-Z water channel (seeCalamita, 2000); RetS (see Goodman et al., 2004; and Zolfaghar et al.,2005); members of the 7TMR-DISM family (see Anantharaman et al., 2003);OprM (see Wong et al., 2000; and SEQ ID NO:11); bacterial proteins suchas OprJ (SEQ ID NO:12), OprN (SEQ ID NO:13), AprF (SEQ ID NO:14), OpmM(SEQ ID NO:15), OpmA (SEQ ID NO:16), OpmD (SEQ ID NO:17), OpmE (SEQ IDNO:18), OpmQ (SE ID NO:35), OpmB (SEQ ID NO:36), OpmJ (SEQ ID NO:37),OpmG (SEQ ID NO:38), OpmJ (SEQ ID NO:39), OpmH (SEQ ID NO:40), OpmK (SEQID NO:41), OpmN (SEQ ID NO:42), OpmF (SEQ ID NO:43), or OpmL (SEQ IDNO:44); OprD family of porins (see Tamber et al., 2006); ACE, or the E.faecalis OG1RF encoded ACE gene (see Sreedhar et al., 2000; and Rich, etal., 1999); PA-IL and PA-IIL galactose- and fucose-binding lectins (seeMitchell et al., 2002); plant and animal virulence genes described by Heet al., 2004; extracellular pyrophosphate moieties (see Bonev et al.,2004); metalloproteases (see Rudner et al., 1999); and transposonencoded surface molecules (see Jacobs et al., 2003).

Other non-limiting examples of virulence factors targeted by a disclosedengineered hmw bacteriocin include those encoded by the open readingframes (ORFs) disclosed in U.S. Pat. No. 6,355,411 and WO 99/27129. Insome embodiments, a factor targeted by a bacteriocin disclosed herein isone encoded by the following ORFs from the U.S. patent:

ORF number Encoding 5 Unknown 9 Unknown 21 Possibly receptor 23 PossiblyABC transporter 33 Unknown 41 Possibly mucin like 43 Unknown 51 Unknown53 Possibly mucin like 85 Unknown 89 Possibly lipoprotein receptor 91Unknown 95 Possibly proteophosphoglycan, cell surface 107 Possibly ABC110 Possibly membrane glycosyltransferase 113 Possibly multidrugresistance protein MexA 132 Possibly muc d 134 Possibly 6-UDP mannosedehydrogenase 149 Possibly MDR transporter potential target 150 Possiblymultidrug resistance protein MexA 203 Possibly ABC transporter ATPasecomponent 204 Possibly ATPase component of ABC transport 205 PossiblyATPase component of ABC transport 206 Possibly ATPase component of ABCtransport 207 Possibly ATPase component of ABC transport 208 PossiblyATPase component of ABC transport 209 Possibly ABC 213 PossiblyNhaP-type Na+/H+ and K+/H+ antiporters 215 Unknown 227 Possibly receptor239 Possibly deoxycytidine triphosphate deaminase 241 Possibly UTPase249 Unknown 255 Unknown 261 Possibly 6-phosphoglyconate dehydrogenase263 Possibly ABC transporter 273 Unknown 277 Possibly PE-PGRS familymember 289 Possibly 6-phosphogluconate dehydrogenase 291 PossiblyGlycosyl transferase 297 Possibly ligA 301 Possibly glycosyltransferase309 Possibly cation/multidrug efflux pump 323 Unknown 327 Unknown 331Possibly sensor with putative PilR kinase 333 Possibly Tonb proteintransport 341 Possibly Pil R 349 Possibly Pil A or R 363 Possibly orfz365 Possibly ABC transporter 375 Possibly mucin 377 Possibly fimT pilus381 Possibly H1 immobilization antigen 383 Possibly fimU 387 PossiblyPilV pilus 393 Possibly pilW et 401 Possibly pil X 403 Possibly antigencd3 411 Unknown 413 Unknown 419 Possibly pil E 421 Possibly pyl y2 427Possibly PE-PGRS outer membrane antigen 437 Possibly ABC ligA

DETAILED DESCRIPTION OF MODES OF PRACTICING THE DISCLOSURE

General

Hmw bacteriocins have the ability to quickly kill bacteria. A few earlyreports of in vivo studies have shown that they can be effective in micefor this application (Haas et al., 1974; Merrikin and Terry, 1972). Thisinvention provides that when administered preferably eitherintraperitonealy or intravenously, wild type R2 pyocin can rescue micefrom acute, lethal peritonitis caused by antibiotic-resistantPseudomonas aeruginosa and that R2 pyocins can act at very low doses,such as 10⁹ pyocins or less than 1 μg total protein in a single dose(data not shown).

For hmw bacteriocins to be clinically useful as antibacterial agents,however; the problem of their narrow bactericidal spectra must beaddressed. While this can be viewed as an advantage in that it ispossible to specifically target a particular species or strain withoutaffecting the normal flora and thereby causing minimal collateraldamage, the types of species/strains that are sensitive to knownbacteriocins are limited. For example, R-type pyocins currently areknown to be produced by some Pseudomonas aeruginosa strains, and haveactivity against a narrow range of other Pseudomonas strains and a fewother gram negative species. R-type bacteriocins from other species havebeen reported (such as Erwinia, see Jabrane 2002, and Yersiniaenterocolitica, see Strauch) but the occurrence appears to be limited.Myoviridae phages, on the other hand, are quite widespread and commonand are found throughout both the gram negative and gram positivebacterial classes.

This disclosure demonstrates that it is possible to change the spectrumof a hmw bacteriocin. A major spectrum determinant among both pyocinsand their related phages lies in the tail fiber, which binds to thebacterial surface specifically, interacting through its C-terminalportion (RBD) with a component of the LPS or other cell surfacestructure. The LPS can be highly variable between different species andstrains of bacteria, and bacteriophage tail fibers are themselves highlyvariable, particularly in this C-terminal region that interacts with thecell surface (Tetart, Desplats). This variability apparently reflectsphages' constant adaptations to changing host surfaces. It has beenobserved that different phage types that infect the same host (E. coliphages P2, Mu, and P1) have sequence similarity in the C-terminalportion of the tail fiber (Haggard-Ljungquist E, Halling C, CalendarR.), indicating that horizontal transfer in these genetic regions likelyplays a role in host specificity. For example, R2 pyocin has a very highdegree of sequence similarity to Pseudomonas phage phiCTX, a phage thatis also very closely related to E. coli phage P2. Comparing the tailfiber sequences of the R2 pyocin and P2, more sequence similarity isseen at the N-terminus (BPAR) than with the C-terminus (RBD), suggestingthat the C-terminus plays the role in host specificity.

As disclosed herein, it is possible to alter the target spectrum of apyocin or other hmw bacteriocin by engineering the C-terminal portion ofthe tail fiber gene. It is notable that this spectrum change can occuracross species and genus barriers, demonstrating that natural R-typepyocins and other natural hmw bacteriocins can be modified as disclosedherein and developed into antimicrobials with broader spectra.

Modified Hmw Bacteriocins

The disclosure provides engineered hmw bacteriocins with altered bindingspecificities and/or affinities. In some embodiments, an hmw bacteriocinof the disclosure specifically binds to exposed surface molecules thatact as virulence factors or fitness factors of pathogenic bacteria. Theterm “specifically (or selectively) binds” refers to a binding reactionthat is determinative of the presence of the bound ligand, often in aheterogeneous population of proteins and other biological matter. As aresult, the engineered hmw bacteriocin once bound specifically cangenerically kill the pathogenic bacteria. Furthermore, in order tobecome resistant to the engineered hmw bacteriocin, the targetedpathogenic bacteria must lose its recognition or binding site for thehmw bacteriocin. Stated differently, if the modified hmw bacteriocinspecifically and exclusively uses the virulence or fitness factor as itsreceptor, the bacteria would be forced to compromise or even completelylose its virulence or fitness in order to escape killing by theengineered hmw bacteriocin.

A modified hmw bacteriocin of the disclosure resembles a bacteriophagetail but comprises a binding capability, or receptor binding domain(RBD), that has been changed relative to an unmodified, naturallyoccurring, or native bacteriocin. The RBD may be changed in amino acidsequence by use of recombinant DNA techniques as described herein. Theterm “recombinant”, typically used with reference to a cell, or nucleicacid, protein, or vector, indicates that the cell, nucleic acid, proteinor vector, has been modified by the introduction of a heterologousnucleic acid or protein or the alteration of a native nucleic acid orprotein, or that the cell is derived from a cell so modified. So arecombinant cell expresses genes that are not found within the native(non-recombinant) form of the cell or expresses native genes that areabnormally expressed, under expressed, or not expressed at all.

In many embodiments, the RBD may be modified to be that of a tail fiberor tail spike from another bacteriocin or a bacteriophage. As onenon-limiting example disclosed herein, the RBD of R2 pyocin is modifiedby fusing the C-terminal portion of the tail fiber protein (RBD) of aphage (that infects a different host) to the N-terminal portion (BPAR)of the R2 tail fiber protein. By fusing the C-terminus of the P2 tailfiber to the R2 PRF15 and co-expressing the P2 cognate chaperone, thetarget bacteria spectrum of the R2 was changed to kill E. coli C. SeeFIG. 2.

In additional embodiments, hmw bacteriocins are engineered otherwise.The disclosure includes an hmw bacteriocin designed or selected torecognize, or target, a surface molecule of a bacterium (such as apathogenic bacterium). The surface molecule may be considered a receptoron a bacterium recognized, or bound, by the hmw bacteriocin.

The disclosure is based on the properties of an hmw bacteriocin tailfiber to bind to, or interact with, a receptor to form a binding pair.The binding or interaction occurs through the RBD of the tail fiber,which is the first member of the binding pair, with the receptor beingthe second member of the pair. In many embodiments, the receptor is abacterial cell surface molecule or portion thereof. In otherembodiments, the receptor is a molecule with properties of a virulenceor fitness factor of a pathogenic bacterium.

A modified or engineered hmw bacteriocin disclosed herein comprises atail fiber having both a base plate attachment region (BPAR) and amodified, or heterologous, RBD. As described herein, the tail fiber is atrimeric structure of three tail fiber protein subunits, each of whichalso comprises a first domain corresponding to, and forming, the BPAR ina tail fiber and a second domain corresponding to, and forming, amodified or heterologous RBD in a tail fiber.

Typically, “heterologous” when used with reference to portions of aprotein or nucleic acid sequence indicates that the sequence comprisestwo or more subsequences that are not usually found in the samerelationship to each other in nature. For instance, a heterologousprotein indicates that the protein comprises two or more subsequencesthat are not found in the same relationship to each other in nature.“Heterologous” also means that the amino acid or nucleic acid sequenceis not normally found in conjunction with the other sequences or is notnormally contained in the selected plasmid, vector, or host. In otherwords, it is not native to the system for which it is now utilized. Forexample, proteins produced by an organism that is not the wild typesource of those proteins.

So in many embodiments, the disclosure includes an hmw bacteriocin tailfiber protein comprising a BPAR of the protein and a modified, orheterologous, RBD. The BPAR is typically at the N-terminal region of atail fiber protein, while the RBD is typically at the C-terminal region.Other than the modified, or heterologous, RBD, the tail fiber proteinmay be that of any naturally occurring hmw bacteriocin, with a pyocin,monocin, enterocoliticin, or meningocin being non-limiting examples. Insome embodiments, the tail fiber protein of R1-pyocin, R2-pyocin,R3-pyocin, R4-pyocin, and R5-pyocin, as represented by SEQ ID NO:1, 3,5, 7, 9, respectively, may be used as described herein. In additionalembodiments, the tail fiber protein may be that or those of the ΦTXphage SEQ ID NO:45, or that of phage PS17 SEQ ID NO:19 or that of theVHML bacteriophage SEQ ID NO:21 and 22.

Embodiments of the disclosure include combinations of an hmw bacteriocintail fiber protein BPAR and a RBD from a bacteriophage tail fiberprotein, as shown in FIG. 3. In some cases, a combination may includethe N-terminal amino acids from position 1 to about position 164 orposition 240 of a bacteriocin tail fiber protein. This polypeptidefragment may be fused to a region of a bacteriophage tail fiber proteinincluding its C-terminal portion containing an RBD. The region may be apolypeptide fragment lacking the N-terminal region from position 1 toabout position 150, about position 170, about position 190, aboutposition 290, about position 300, or about position 320.

Using the R2 pyocin and the P2 phage tail fiber protein as non-limitingexamples, the BPAR containing fragment may include the N-terminal aminoacids from position 1 to position 164 or 240. See FIGS. 4-7. The RBDcontaining fragment may include the C-terminal, and from about 347 toabout 755 amino acids in length of the P2 or related phage tail fiberproteins. The fusion may be readily prepared by recombinant DNAtechniques with nucleic acid sequences encoding the R2 tail fiberprotein, such as prf15, and the P2 phage gene H encoding its tail fiberprotein. When the RBD is derived from the tail fiber of another hmwbacteriocin or myoviridae, the cognate chaperone of the RBD needs to beco-expressed with the fusion tail fiber genes in order to ensure theassembly of the modified tail fibers into a functioning pyocinstructure. See FIG. 8.

Another non-limiting example is the use of the R2 pyocin and the phiV10phage. The BPAR containing fragment may include the N-terminal aminoacids from position 1 to position 161 or 164 of the PRF 15 protein. TheRBD containing fragment may include the c-terminal amino acids fromposition 204, 211, or 217 to position 875 of the V10 tail spike protein.See FIG. 11A, and SEQ ID NOs: 67, 68, 69.

In other embodiments, a modified RBD comprises a change in the aminoacid sequence of the RBD relative to a naturally occurring RBD orrelative to the BPAR present in the tail fiber protein. Non-limitingexamples of a change in amino acid sequence include substitution,insertion (or addition), or deletion of one or more amino acids.

In embodiments comprising the substitution of RBD amino acid residues,about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%,about 22%, about 24%, about 26%, about 28%, about 30%, about 35%, about40%, about 45%, or about 50%, or more, of the C-terminal in a tail fiberprotein are substituted. In some embodiments, the substitutions arewithin about 245, about 260, about 275, or about 290, or more, residuesfrom the C-terminal.

The positions for substitution may be any one or more, in anycombination, within that region. Exemplary positions include, but arenot limited to, 448, 449, 452, 453, 454, 455, 459, 460, 462, 463, 464,469, 472, 473, 474, 475, 478, 480, 484, 485, 486, 491, 494, 496, 497,498, 499, 505, 506, 507, 508, 510, 512, 514, 517, 518, 519, 520, 521,523, 527, 528, 530, 531, 533, 535, 537, 538, 541, 543, 546, 548, 561,603, 604, 605, 606, 610, 618, 621, 624, 626, 627, 628, 629, 631, 632,633, 638, 641, 642, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654,655, 657, 659, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673,674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687,688, 689, and 691, as well as any combination thereof, in SEQ ID NO: 1,3, 5, 7, or 9. In some embodiments, the substitution is conservative asdescribed herein. In other embodiments, the substitution is with anon-conservative substitution.

In further embodiments, insertions and deletions of amino acid residueswithin the same region at the C-terminal of a tail fiber protein may bemade.

Other sources of RBDs include, but are not limited to, T-4 and otherT-even or pseudoT-even phages, phages T-3 and T-7, T-7 super-group ofphages, phage Mu, phage P22, phage L-413c, podophages, lambdoid phagesand even polysaccharide binding or specific protein binding enzymes orligands, the binding properties of which can serve RBD functions as didthe tail spike protein of phiV10.

RBD from Diversification

In further embodiments, a tail fiber protein comprises a substitutionwith, or insertion of, an RBD derived from an organism that diversifiesthe structure by deploying a Diversity Generating Retroelement (DGR), asdescribed in published Patent Application US 2006-0121450, publishedJun. 8, 2006 (incorporated herein by reference as if fully set forth).The major tropism determinant (MTD) of Bordetella bacteriophage BPP-1 isone such structure. The sequence of MTD is represented by SEQ ID NO:24as disclosed herein. In other embodiments, the substitution is with partof the MTD sequence, such as, but not limited to, the region fromresidue 49 to 381, from residue 171 to 381, or from residues 306 to 381,of SEQ ID NO:24. The insertion of the MTD sequence, or any fragmentthereof (such as those listed above), to the end of a tail fiberprotein, such as after position 691 of SEQ ID NO:3, is within theembodiments disclosed herein. The substitution of the MTD sequence, orany fragment thereof (such as those listed above), may be for anynon-BPAR region of a tail fiber protein. Non-limiting examples includethe region of SEQ ID NO:1, 3, 5, 7, or 9 beginning at about position643, 625, 562, 448, 428, 231, and 163 through to the C-terminus of thesequence (see FIGS. 4-7 for exemplifications of these substitutions).

As described herein, the tropism determinant sequence in a tail fibermay be diversified to produce a plurality of modified or heterologousRBDs. The nucleic acid sequence encoding the tropism determinantcomprises a variable region (VR) which may be operatively linked, in cisor in trans, to a template region (TR) such that the TR is a templatesequence that directs site-specific mutagenesis of the VR. The operativelinkage of the VR and TR regions also includes an operative linkage tosequences encoding a reverse transcriptase (RT) activity, which may bepresent in trans relative to the VR. Sites of variability in the VR ofthe tropism determinant correspond to adenine residues in the generallyhomologous template region, TR, which itself is invariant and essentialfor sequence alterations in the VR. So while an initial molecule maycontain a TR that is identical to the VR, the adenine residues presentin the TR will result in the mutagenesis or diversification of thecorresponding positions in the VR sequence. So if the TR sequence is aperfect direct repeat of the sequence in the VR, diversification of theVR region results in one or more adenine residues in the VR, also foundin the TR, being mutated to another nucleotide, that is cytosine,thymine or guanine, without change in the TR sequence. This system maybe used to alter the VR region, and thus the RBD, of a modified tailfiber protein as described herein.

Upon diversification, the tail fiber protein may be varied such that theresultant RBD has at least 80%, at least 85%, at least 90%, or at least95% homology to the major tropism determinant (MTD) of Bordetellabacteriophage BPP-1, as represented by SEQ ID NO:24. As describedherein, the tail fiber protein and tropism determinant combination maybe a substitution, or an insertion, of a tropism determinant sequence orportion thereof into the tail fiber protein sequence. Thus the resultanttail fiber protein may be viewed as comprising a substitution orinsertion with a binding domain with at least 80%, at least 85%, atleast 90%, or at least 95% homology as recited above.

A nucleic acid molecule encoding a tail fiber and a tropism determinantcombination may be used for diversification and sequence variation. Thusnucleic acid combinations of sequences encoding all or part of a tailfiber protein, and all or part of a tropism determinant, are within thedisclosed embodiments. Other embodiments include nucleic acid moleculesencoding any tail fiber protein with a modified or heterologous RBD asdisclosed herein. In some embodiments, the encoded modified orheterologous RBD comprises a change in the amino acid sequence of theRBD relative to a naturally occurring RBD or relative to the BPARpresent in the tail fiber protein as described above.

In additional embodiments, a tail fiber protein encoding nucleic acidmolecule may be made available for diversification to form a modifiedtail fiber protein disclosed herein. The nucleic acid molecule, undercontrol of a suitable promoter, is operatively placed 5′ to anatd-TR-brt region. The TR sequence may be referred to as TR′ andprepared based upon the VR sequence as discussed below. The resultingnucleic acid construct may carry a deletion of the transcriptionterminator structure upstream of the atd.

A region of the nucleic acid molecule encoding the C-terminal end of thetail fiber protein as described above, is selected to be the VR and thenoperatively linked to a TR′ sequence containing adenine residues atpositions, that when varied, direct amino acid changes in the sequenceencoded by the VR. Such adenine residues may be deliberately designed tobe the first or second position of codons within the VR. The TR′sequence can initially be identical to the selected VR followed by sitedirected mutagenesis or de novo nucleic acid synthesis to prepare a TR′sequence that contains adenine residues only at the correspondingpositions to direct mutagenesis and diversification in the encoded tailfiber protein.

Preparation and Use of Hmw Bacteriocins

The nucleic acid molecules described herein may be used to express andprepare tail fiber proteins, including modified or engineered proteins,by any means known to the skilled person. In some embodiments, theexpression is via the use of a vector containing the nucleic acidmolecule operably linked to a heterologous promoter that can direct theexpression of the encoded tail fiber protein. The promoter can becontrolled by an exogenous molecule that acts as an inducer orco-repressor to express or not express the tail fiber proteins.

In many embodiments, the expression may occur with expression of anaccessory gene, such as a tail fiber “chaperone” encoding sequencereported for various bacteriophages. The presence of a cognate chaperonefor the RBD portion when derived from a tail fiber of a hmw bacteriocinor myoviridae facilitates assembly of an hmw bacteriocin of thedisclosure without necessarily becoming a part of the bacteriocin, asshown in FIG. 8. One non-limiting example of a chaperone is encoded byR2 prf16 (SEQ ID NO:4), and it corresponds to (or is the cognatechaperone for) the R2 pyocin tail fiber protein encoded by prf15 (SEQ IDNO:3). Other examples include gene G in the P2 (SEQ ID NO:26), gene G onL-413c (SEQ ID NO:29), the cognate chaperone, SEQ ID NO: 20, for thePS17 tail fiber, and the Orf 38 (SEQ ID NO:23) in VHML bacteriophages,each of which is the cognate chaperone to the respective tail fiber genein each of these myoviridae phages. These genes are homologues to thephage T4 gp38 (SEQ ID NO:32), which is known to be responsible forproper folding of the T4 tail fiber (SEQ ID NO:31) into trimers (Burda,Qu, Hashemolhosseni).

The use of a cognate chaperone is advantageous because a non-cognatechaperone may be insufficient to correctly fold a given tail fiberprotein and/or assemble it into an hmw bacteriocin, as shown in FIG. 8.As a non-limiting example, the R2 prf16 gene product has been observedto be insufficient to complement the folding of a modified tail fibercompromising an R2 BPAR fused to a P2 RBD portion of a tail fiber.Without being bound by theory, and offered to improve the understandingof the present disclosure, it is believed that a chaperone may actspecifically on the C-terminal portion of its cognate tail fiber proteinand that the tail fibers and their chaperones have co-evolved. However,Qu et al. isolated a T4 gp37 tail fiber mutant that suppresses therequirement for gp38, its cognate chaperone. This mutant had in gp37aduplication of a coiled-coil motif, which may itself play a role infolding. Therefore, it is further believed that a tail fiber protein maybe designed to contain such a change so that it folds properly withoutthe need to co-express a cognate chaperone.

Therefore, embodiments of the disclosure include a bacterial celltransfected with a nucleic acid molecule encoding a modified orengineered tail fiber protein, optionally co-expressed with a chaperone,as described herein. Expression of the nucleic acid molecule, optionallywith an accessory (chaperone) protein, results in the production ofmodified or engineered tail fibers of the disclosure. The disclosurealso includes expression of more than one modified or engineered tailfiber protein through the use of more than one nucleic acid molecule toresult in mixed homotrimeric tail fibers or even heterotrimeric tailfibers. Additionally, sequences encoding the tail fiber protein andchaperone may be contained within a single nucleic acid molecule, suchas a plasmid or other vector, or by separate molecules. Where a singlenucleic acid molecule is used, the sequences optionally may be under thecontrol of the same regulatory sequence(s). Alternatively, the codingsequences may be under separate regulatory control.

In some embodiments, the bacterial cell is also capable of expressingthe additional subunits to form an hmw bacteriocin comprising a modifiedor engineered tail fiber. In one group of embodiments, the endogenoustail fiber protein coding sequence of the bacterial cell is inactivatedor deleted. Optionally, the other subunits may be encoded by sequenceson a nucleic acid molecule, such as a plasmid or other vector, separatefrom that which contains a sequence encoding a tail fiber protein and/orchaperone. Thus the tail fiber protein and/or chaperone may be providedone or more nucleic acid molecules in trans relative to the othersubunits.

The nucleic acids, vectors, and bacterial cells may be used in a methodof producing a modified or engineered hmw bacteriocin as disclosedherein. Such a method may comprise culturing bacterial cells containingnucleic acid molecules as described above under conditions resulting inthe expression and production of the tail fiber and hmw bacteriocin. Insome embodiments of the disclosure the conditions are in vivo within ananimal.

In one group of embodiments, a method of preparing an hmw bacteriocincomprises expressing the bacteriocin subunits, including the modified orengineered tail fiber protein, in a host bacterium, and harvesting thehmw bacteriocin from the bacterial culture. The host bacterium is acomplementary host production bacterium that encodes and expresses theother subunits necessary for the production of the bacteriocin. The term“host bacterium” or “host bacteria” refers to a bacterium or bacteriaused to produce an hmw bacteriocin disclosed herein. Host bacteria orbacterium may also be referred to as “host production bacterium”, “hostproduction bacteria”, “production bacterium” or “production bacteria”.The “harvesting of an hmw bacteriocin from a bacterial culture”generally comprises removing the bacteriocin from the host bacterialculture.

In an alternative group of embodiments, a method of preparing an hmwbacteriocin with a modified tail fiber as described herein is provided.The method may comprise preparing a nucleic acid molecule encoding amodified tail fiber protein by any means disclosed herein and expressingthe nucleic acid molecule in a cell under conditions wherein an hmwbacteriocin is produced.

Embodiments of the disclosure include an hmw bacteriocin comprising atail fiber protein as described herein. In one group of embodiments, thebacteriocin comprises a tail fiber protein comprised in part of theamino acid sequence represented by SEQ ID NO:1, 3, 5, 7, 9. In otherembodiments, the bacteriocin is a modified or engineered pyocin,monocin, enterocoliticin, or meningocin comprising a tail fiber with aheterologous modified RBD. In many embodiments, the heterologousmodified RBD binds a bacterial virulence or fitness factor.

In further embodiments, engineered hmw bacteriocins with multivalenttail fibers are disclosed. MTD of Bordetella bronchisepticabacteriophage BPP-1 has been found by X-ray crystallographic analysis tobe a highly intertwined pyramidal homotrimer with the three sets oftwelve non-contiguous variable amino acid residues forming three ratherflat receptor-binding sites at the tetrahedron's base and located in aconvergently evolved C-type lectin (“CTL”) domain. Comparison of thestructures of five MTD variants at 1.5 Å resolution showed that the mainchain conformation of variable residues is structurally invariant, withinserts in the CTL and trimeric assembly both contributing to formationof a static scaffold for combinatorial display of variable residues,thereby minimizing the incidence of protein misfolding (McMahon et al.,2005). Thus a single tail fiber may be generated to contain threeproperly folded mixed monomers, since the structures of the varianttropism determinant fibers are identical except for the non-interacting,solvent-exposed twelve amino acid residues.

The structure of the dominant MTD-P1 variant bound to its receptor, theBordetella virulence factor pertactin, also has been solved bycrystallography and characterized. One of the monomers of MTD binds toone structural domain on pertactin; a second identical monomer of thesame MTD binds a different, non-symmetrical structural domain of thesame (monomeric) pertactin molecule; a third MTD monomer remainsunbound.

The above variant MTD structures and the binding interaction between MTDand its target, pertactin, may be applied to the design and selection ofmultivalent tail fibers. For example, it is evident that an MTD monomercan exhibit affinities for two different structural domains and yet inmultimeric format possess sufficient avidity to effect functional phagebinding and infection. Furthermore, not all monomers of a fiber need bebound to a receptor to provide adequate avidity for phage binding andinfection. These data and conclusions along with the knowledge that forat least T4 bacteriophages, also a member of the myoviridae family, onlythree (homotrimeric) tail fibers need be bound to receptors to triggertail sheath contraction and core penetration of bacterial membranes,indicates several means of generating a multivalent hmw bacteriocin.

Such engineered multivalent hmw bacteriocins have broader host rangesand are capable of binding to more than a single virulence or fitnessfactor even on the same bacterial organism, thereby making it moredifficult for targeted bacteria to develop resistance by mutational lossof expression of all targeted, relevant receptors. An R-type bacteriocincan be engineered to possess two independent sets of three identicaltail fibers. The fibers of one set comprised of the same threenon-identical monomers, and the fibers of the other set comprised ofthree different non-identical monomers. Each monomer can possess bindingaffinities for two different epitopes (e.g. two different receptors),just as does the tropism determinant. Thereby any bacterium expressingany one or more of the 12 different targeted receptor molecules (2“epitopes”/monomer times 3 monomers/tail fiber times 2 sets of differenttail fibers/R-type bacteriocin equals 12 targeted receptors) would bindthe engineered multivalent hmw bacteriocin and trigger its penetrationof the membrane. Such engineered hmw bacteriocins have an unnaturallybroad host range and, in addition, make it highly unlikely that abacterium expressing more than a single targeted receptor could becomeresistant to the engineered hmw bacteriocins.

In other aspects, methods for the use of an hmw bacteriocin of thedisclosure are provided. In some embodiments, a method of compromisingthe integrity of the cytoplasmic membrane of a bacterium is disclosed.The method may comprise contacting a target bacterium with a fusionpeptide or an hmw bacteriocin, or portion thereof, as disclosed herein.Alternatively, the contact may be with an hmw bacteriocin containingcomposition disclosed herein.

In one group of embodiments, the contacting occurs in vivo within asubject. Thus a method of compromising the membrane integrity of abacterium in a subject is disclosed. The method may compriseadministering a fusion peptide or an hmw bacteriocin or a portionthereof as described herein to the subject. In another group ofembodiments, the contacting occurs ex vivo or in vitro.

The methods can be used as a stand-alone therapy or as an adjunctivetherapy, such as for the treatment of bacterial populations. Numerousantimicrobial agents (including antibiotics and chemotherapeutic agents)are known which would be useful in combination with these methods totreating bacteria-based conditions.

In yet additional embodiments, a method of forming non-virulent or unfitbacteria progeny from virulent progenitor bacteria is provided. Themethod may comprise contacting virulent bacteria with an hmw bacteriocinwhich binds a virulence or fitness factor of said virulent progenitorbacteria as disclosed herein. The method then may continue by allowingselection of non-virulent bacteria progeny that no longer express thevirulence or fitness factor.

In an alternative embodiment, a method of maintaining a population ofnon-virulent bacteria is provided. The method may comprise contactingthe population with an hmw bacteriocin which binds a virulence orfitness factor of virulent bacteria. The method then continues andprevents propagation of virulent bacteria. Without being bound bytheory, and offered to improve the understanding of the disclosure, anemergence of bacterial resistance to an engineered hmw bacteriocin willbe accompanied by a compromised virulence or fitness of the pathogenicbacteria.

The methods of the disclosure also may be applied in an environmentwhere bacterial growth is not desired or is considered to be harmful.Non-limiting examples include the sterilizing of environments, includingmedical settings and operating room facilities; as well as food or foodpreparation surfaces or areas, including where raw meat or fish arehandled or discarded. The methods may also be used to sterilize heatsensitive objects, medical devices, and tissue implants, includingtransplant organs.

In particular, food or food products are affected by pathogenic orundersirable bacteria, such as certain strains of E. coli. However, insome instances, only certain species or groups of bacteria arepathogenic, so specific bacteriocin can be designed to target thesegroups or species. For example, one may choose to kill or mitigate an E.coli strain, such as O157:H7, but leave other natural, non-harmful E.coli unaffected. Therefore, selective or whole, santization orsterilization of bacteria is possible depending upon the use of one ormore bacteriocins.

In another embodiment, methods of diagnostic screening or selection areprovided. A sample of a suspected or known bacteria can be screenedagainst one or more engineered bacteriocins to identify their potentialtherapeutic effects against the bacteria.

Furthermore, the engineered bacteriocins can be utilized to selectivelyor generally detect the presence of the pathogenic bacteria. In someinstances, the bacteriocins would be labeled with a detectable marker,such that in the presence of the targeted bacteria, the label would bedetected or identified.

Target Bacteria

The engineered hmw bacteriocins of the disclosure may be modified totarget a receptor on a variety of bacterial species and strains,including pathogenic bacteria, such as nosocomial or pyogenic bacteria,as non-limiting examples. In addition to targeting the virulence factorsof select bacteria as described herein, bacteria that are alreadysusceptible to bacteriophages are one non-limiting group of bacteriathat may be inhibited by an hmw bacteriocin, such as an engineeredpyocin, of the disclosure. These bacteria include the gram negativebacteria that are susceptible, as well as not susceptible, to naturallyoccurring pyocins. Additional non-limiting examples include gramnegative bacteria as a group as well as gram positive bacteria. Thereare reports of hmw bacteriocin-like entities in gram positive bacteriathat target other gram positive bacteria (Thompson & Pattee, 1981;Birmingham & Pattee, 1981; Zink et al., 1995). In some embodiments, thetarget bacterium is identified or diagnosed. Non-limiting examples ofsuch bacteria include those of the genus Escherichia, Staphylococcus,Clostridium, Acinetobacter, Pseudomonas, or Streptococcus.

As a non-limiting example of targeting a virulence factor, thedisclosure includes the use of a phage tail fiber protein RBD like thatof the tail spike protein from the podoviridae phage phiV10 that infectsE. coli O157:H7 but does not infect a mutant strain TEA026 derivedtherefrom that has lost the O157 antigen (Ho and Waldor, 2007). Thebinding of this phage requires the presence of the O157 antigen, avirulence factor, involved in gut colonization by the pathogenic E. coliO157:H7 organism (Ho and Waldor, 2007). Therefore, an hmw bacteriocin ofthe disclosure may contain a modified tail fiber protein containing theglobular RBD from the tail spike protein (SEQ ID NO:60) of the abovedescribed phage phiV10 such that the modified hmw bacteriocin targets avirulence factor, the 0157 antigen, of E. coli O157:H7. The globulartail spike protein does not have a cognate chaperone as it apparentlyfolds without such, and thus a chaperone is not required for theassembly of its fusion with the BPAR of R2 Prf15.

An “infection” refers to growth of bacteria, such as in a subject ortissue or non-bacterial cell, wherein the bacteria actually orpotentially could cause disease or a symptom in the subject, tissue ornon-bacterial cell. Treatment of an infection may include prophylactictreatment of substances or materials. Non-limiting examples includedonated organs, tissues, and cells; medical equipment, like a respiratoror dialysis machine; or wounds, such as those during or after surgery.Other uses include the removal of target bacteria which may causeproblems upon further growth. In additional embodiments, an hmwbacteriocin is used to treat food, plants or harvested parts of plantswith bacterial infections or contaminations, or to treat environmentaloccurrences of the target bacteria, such as in a hospital or commercialsetting.

The disclosure provides for the treatment, by administration or contactwith an hmw bacteriocin disclosed herein to target the bacteria, of suchinfections in tissues and subjects as follows. The infections includethe common infections of the cornea (“keratitis” and corneal ulcers), atleast two-thirds of which are caused by P. aeruginosa. Approximately 30%of these pathogens are reported to be resistant to multiple antibiotics(Mah-Sadorra et al., 2005). Bacterial infection of the cornea isconsidered a relatively uncommon, but serious condition, requiringurgent medical attention, because of the potential for reduced vision oreven vision loss in the affected eye(s). Other common infections whichmay be treated, and are caused by antibiotic-resistant P. aeruginosa,include ear infections, e.g. “swimmer's ear” (Roland & Stroman, 2002),those secondary to severe burns and wounds (Holder, 1993), and cysticfibrosis. Cystic fibrosis is consistently aggravated by chronic,antibiotic-resistant infections caused by P. aeruginosa and its closerelative, Burkholderia cepacia (Govan & Deretic, 1996), and thesepathogens in cystic fibrosis may be treated by use of an engineered hmwbacteriocin. Because bacteriocins like pyocins will toleratefreeze-drying (Higerd et al., 1969), the disclosure includes afreeze-dried formulation of a bacteriocin for administration to enhancethe likelihood of successful delivery to the upper and/or lower airwayof the respiratory tract.

As described herein, the treatment of a subject is typically of “asubject in need of treatment”. The determination, or diagnosis, of theneed for treatment may be made by a skilled person, such as a clinician,by use of art recognized means. In some embodiments, the subject is ananimal or plant with a bacterial infection that is potentiallylife-threatening or that impairs health or shortens the lifespan of theorganism.

In additional embodiments, a method to kill or inhibit the growth ofbacteria in a biofilm is provided. Such a method may comprise contactinga biofilm with an hmw bacteriocin disclosed herein which targetsbacteria in the biofilm.

As described herein, an anti-bacterial hmw bacteriocin is used toinhibit growth, survival, or replication of a particular bacterium. Thebacterium may be a pathogenic or environmentally deleterious strain, ormay be treated in a prophylactic manner. A pathogenic microorganismgenerally causes disease, sometimes only in particular circumstances.

The bacteria may also be that of a nosocomial (hospital derived)infection, environmental bacteria, and pyogenic (pus forming) bacteria.The methods and compositions of the disclosure can be used to inhibitgrowth of nosocomial bacteria, including bacteria that populate atypical hospital environment, or bacteria that are present on human skinor nose or in the human gastrointestinal tract, or bacteria that infectand form pus in wounds. Nosocomial infections are infections whichbecome evident during a hospital stay or are related to a procedureperformed in a hospital. These procedure-related infections often becomeevident after patients are discharged from the hospital. The most commonnosocomial bacterial infections are urinary tract infections,surgical-site infections, pneumonia, C. difficile associated diarrheaand pseudomembrane colitis, and serious systemic infections in whichbacteria can be grown from blood.

The methods and compositions of the disclosure may be used to inhibitgrowth of gram negative or gram positive bacteria. Non-limiting examplesof gram positive bacteria include Staphylococcus (pyogenic),Enterococcus (opportunistic), Streptococcus, Enterococcus, Bacillus,Micrococcus, Mycobacterium, Corynebacterium, and Clostridium.Non-limiting examples of gram negative bacteria include Pseudomonas(pyogenic), E. coli (opportunistic), Salmonella (opportunistic),Campylobacter (opportunistic), Proteus (pyogenic), Klebsiella(opportunistic), Enterobacter (pyogenic), Citrobacter (pyogenic), gramnegative non-fermenter rods (such as Acinetobacter), and Shigella. Thepyogenic cocci are spherical bacteria that cause various suppurative(pus-producing) infections in animals. Included are the gram-positivecocci Staphylococcus aureus, Streptococcus pyogenes, and Streptococcuspneumoniae, and the gram-negative cocci, Neisseria gonorrhoeae, and N.meningitidis.

In additional embodiments, the disclosed methods and compositions of thedisclosure are used to inhibit growth, particularly of antibioticresistant bacteria. Non-limiting examples include numerous bacterialpathogens that have become multi-drug resistant (MDR).

Engineering Pyocins

Francois Jacob discovered and first described pyocins as high molecularweight bacteriocins (Jacob, 1954). Similar bacteriocin-like entitieshave been described in multiple other gram negative bacteria (Coetzee etal., 1968) as well as in Listeria moncytogenes (Zink et al. 1995) andStaphylococcus aureus (Thompson and Pattee, 1981), both of which aregram positive organisms. While pyocins morphologically resemble thetails of contractile (myoviridae) bacteriophages, they are not simpledefective phages; there are meaningful differences. For example,differences exist in physical and chemical stability between pyocins andphage tails (Kageyama & Egami, 1962; Nakayama et al., 2000). While thehost ranges of pyocins are relatively narrow and usually restricted tostrains of the same species, there are exceptions (Morse et al, 1976;Blackwell et al., 1982). On the other hand, myoviridae bacteriophagescan exhibit broad host ranges, and their host ranges, like those ofR-type pyocins, are determined by the binding specificities of the tipsof their tail fibers (Tetart et al., 2001).

For numerous phage tail fibers, the distal (3′-terminal) third of thegene varies in mutants or variants with altered phage host ranges, or“tropisms” (Ackermann, 2003). As a non-limiting example, the majortropism determinant (MTD), the receptor binding protein of Bordetellabacteriophage BPP-1, varies greatly in sequence (Liu et al., 2004;Doulatov et al. 2004). Variation in tropism determinants depends on aphage-encoded retroelement (Diversity Generating Retroelement, or DGR)that belongs to a family of DGRs implicated in generating sequencevariation in various phage and bacterial genomes. The Bordetella DGR canproduce more than 1013 different sequence variants of MTD, rivaling the10¹⁴-10¹⁶ possible sequences of antibodies. Tropism determinant variantsare produced by a unique adenine-specific mutagenesis process involvingDGR-encoded reverse transcriptase (bRT) and a stable template region(TR). Variability in MTD is focused to 12 adenine-encoded amino acidsthat are scattered across its C-terminal variable region (VR) (Doulatovet al. 2004). The 3-dimensional crystal structures of numerousBordetella MTD variants have been solved and confirm, as describedbelow, that the tip of the structure determines the binding specificityand thereby the major tropism (host range) of the phage (McMahon et al.,2005). Thus, as further described below, the tropism determinant and itsrelated DGR system may be used in the practice of the disclosure.

Many Pseudomonas species possess the genes for the R-type pyocins(Takeya et al., 1969; Kageyama, 1975). The R-type pyocin locus consistsof about 16 complementation groups including about 10 structural genesplus regulatory and chaperone genes (Shinomiya et al. 1983a; Shinomiyaet al., 1983b). Morphologically and genetically the R-type pyocinsresemble the tails of myoviridae bacteriophages but have no headstructure and thus no nucleic acid content (Kageyama, 1964; Ishii etal., 1965; Shimizu et al., 1982). They are thought to have evolved fromthe phage tail structure of a P2-related ancestor, but they are notsimple defective phages, having been further adapted for their role asdefensive bactericidal agents (Nakayama et al, 2000). Similar tobacteriophages, however, pyocins do bind to specific molecular“receptors” on target bacteria and penetrate their membranes with a“core” or needle-like structure (Uratani & Hoshino, 1984). As animmediate consequence of the core penetration of the membranes, thebacterium is killed by compromise of the integrity of its cytoplasmicmembrane and dissipation of its membrane potential, a bactericidal eventthat can result from an attack by a single pyocin (Iijima, 1978; Uratani& Hoshino, 1984; Strauch et al., 2001).

The RBD, or Receptor Binding Determinant of R-pyocin binding, of atypical R-type pyocin binds to a bacterial surface molecule. In the caseof an R2 pyocin isolate, the RBD resides in the carboxy-terminal portionof its tail fiber. The tail fiber is a homotrimer of the product of theprf15 gene (Nakayama et al., 2000). Modification of the RBD in the prf15gene and recombination of the modified prf15 gene into a system thatproduces R-type pyocins allows production of an engineered pyocin withmodified binding specificity.

The major tropism determinant (MTD) of Bordetella bacteriophagepossesses several unique and useful properties as a binding domain. Thefunctional form of MTD in Bordetella bacteriophage is a homotrimer thatbinds the virulence factor protein, pertactin, in Bordetella. Thus, theMTD gene may be fused to the distal end of the prf15 gene to takeadvantage of the MTD properties. So as described herein, an aspect ofthe disclosure includes construction of a fusion protein between the P.aeruginosa R-type pyocin tail fiber protein (PRF15) and the majortropism determinant (MTD) of Bordetella phage, BPP-1. A PRF15-MTD fusionmay be used to complement in trans a P. aeruginosa PA01Δprf15 to bindand kill pertactin-expressing Bordetella bronchiseptica orpertactin-expressing E. coli.

Additionally, the P2 or P4 bacteriophage may be used as a surrogate toharbor the prf15-MTD or other tail fiber fusion genes such that thegenotype is coupled to the binding phenotype of the tail fiber. Thispermits efficient transduction, selection, and isolation of the tailfiber gene encoding the desired RBD.

Modes of Administration

An engineered hmw bacteriocin of the disclosure may be administered byany suitable means. Non-limiting examples include topical or localizedadministration as well as pulmonary (inhalation), gastrointestinal, bycatheter or drip tube, or systemic administration to a subject.Representative, and non-limiting, examples of systemic administrationinclude intraperitoneal and intravenous administration. The protectiveeffects of intraperitoneally and intravenously administered pyocins havebeen demonstrated in mice infected systemically with lethal doses P.aeruginosa strains sensitive in vitro to the administered pyocins(Merrikin & Terry, 1972; Haas et al., 1974). In some embodiments,contact between an hmw bacteriocin disclosed herein and a targetbacterial population results in a decrease in the population of at least10, at least 100, at least 1000, or at least 10,000, or more, folddecrease relative to the absence of the bacteriocin. In otherembodiments, the contact may result in a decrease in detectability ofthe bacteria by at least 5, at least 10, at least 20, at least 30, atleast 40, or at least 50, or more, fold relative to the absence of thebacteriocin.

An engineered hmw bacteriocin of the disclosure may be administered toany subject afflicted with, diagnosed as afflicted with, or suspected ofbeing afflicted with, an infection or contamination by bacteriasusceptible to the hmw bacteriocin. Non-limiting examples of such asubject include animal (mammalian, reptilian, amphibian, avian, andfish) species as well as insects, plants and fungi. Representative, andnon-limiting, examples of mammalian species include humans; non-humanprimates; agriculturally relevant species such as cattle, pigs, goats,and sheep; rodents, such as mice and rats; mammals for companionship,display, or show, such as dogs, cats, guinea pigs, rabbits, and horses;and mammals for work, such as dogs and horses. Representative, andnon-limiting, examples of avian species include chickens, ducks, geese,and birds for companionship or show, such as parrots and parakeets. Ananimal subject treated with an engineered bacteriocin of the disclosuremay also be a quadruped, a biped, an aquatic animal, a vertebrate, or aninvertebrate, including insects.

In some embodiments, the subject to be treated is a human child or otheryoung animal which has yet to reach maturity. Thus the disclosureincludes the treatment of pediatric conditions comprising infection withbacteria or other microorganism susceptible to an hmw bacteriocin of thedisclosure.

The disclosure also provides for the treatment or prevention of anopportunistic infection, such as that resulting from an undesirablegrowth of bacteria that are present in the microbial flora of a humansubject or a non-human animal. An opportunistic infection may be theresult of an immunosuppressed condition in a subject or the result ofantibiotic treatment that alter the commensal flora of the genitourinary(GU) or gastrointestinal (GI) tract. Thus the disclosure also providesfor the treatment or prophylaxis of immunosuppressed subjects andsubjects exposed to other pharmaceutical agents. An hmw bacteriocin withits anti-bacterial activity may be used in combination with anotheranti-bacterial or anti-microbial agent, such as an antibiotic oranti-fungal agent as non-limiting examples. An “anti-microbial agent” isan agent or compound that can be used to inhibit the growth of, or tokill, single celled organisms. Anti-microbial agents includeantibiotics, chemotherapeutic agents, antibodies (with or withoutcomplement), chemical inhibitors of DNA, RNA, protein, lipid, or cellwall synthesis or functions.

In some embodiments, an hmw bacteriocin or fusion protein is formulatedwith a “pharmaceutically acceptable” excipient or carrier. Such acomponent is one that is suitable for use with humans, animals, and/orplants without undue adverse side effects. Non-limiting examples ofadverse side effects include toxicity, irritation, and/or allergicresponse. The excipient or carrier is typically one that is commensuratewith a reasonable benefit/risk ratio. In many embodiments, the carrieror excipient is suitable for topical or systemic administration.Non-limiting pharmaceutically suitable carriers include sterile aqueousor non-aqueous solutions, suspensions, and emulsions. Examples include,but are not limited to, standard pharmaceutical excipients such as aphosphate buffered saline solution, water, emulsions such as oil/wateremulsion, and various types of wetting agents. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyloleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like.

Additional formulations and pharmaceutical compositions disclosed hereincomprise an isolated hmw bacteriocin specific for a bacterial host; amixture of two, three, five, ten, or twenty or more bacteriocins thattarget the same bacterial hosts; and a mixture of two, three, five, ten,or twenty or more bacteriocins that target different bacterial hosts ordifferent strains of the same bacterial host.

Optionally, a composition comprising an hmw bacteriocin of thedisclosure may also be lyophilized using means well known in the art.Subsequent reconstitution and use may be practiced as known in thefield.

Also provided are formulations comprising microencapsulated hmwbacteriocin. In some embodiments, these may provide sustained releasekinetics or allow oral ingestion to pass through the stomach and intothe small or large intestine. In general, the pharmaceuticalcompositions can be prepared in various forms, such as granules,tablets, pills, suppositories, capsules (e.g. adapted for oraldelivery), microbeads, microspheres, liposomes, suspensions, salves,pastes, lotions, and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions comprising the therapeutically-active compounds.Stabilizing agents, wetting and emulsifying agents, salts for varyingthe osmotic pressure, or buffers for securing an adequate pH value maybe included.

An hmw bacteriocin is typically used in an amount or concentration thatis “safe and effective”, which refers to a quantity that is sufficientto produce a desired therapeutic response without undue adverse sideeffects like those described above. An hmw bacteriocin may also be usedin an amount or concentration that is “therapeutically effective”, whichrefers to an amount effective to yield a desired therapeutic response,such as, but not limited to, an amount effective to slow the rate ofbacterial cell division, or to cause cessation of bacterial celldivision, or to cause death or decrease rate of population growth of thebacteria. The safe and effective amount or therapeutically effectiveamount will vary with various factors but may be readily determined bythe skilled practitioner without undue experimentation. Non-limitingexamples of factors include the particular condition being treated, thephysical condition of the subject, the type of subject being treated,the duration of the treatment, the nature of concurrent therapy (ifany), and the specific formulations employed.

Additionally, and in anticipation of a possible emergence of bacterialresistance to an engineered hmw bacteriocin, there can be a concomitantcompromise of the organisms' virulence or fitness where the bacteriocintargets the virulence or fitness factor of the targeted bacteria.Because a major, but non-limiting, mechanism by which a bacterium maybecome resistant to an hmw bacteriocin is the loss of its receptor forthe bacteriocin, the targeting of a virulence or fitness factor asdisclosed herein provides many advantages over traditional antibioticsand bacteriophages. The resistance to traditional antibiotics andbacteriophages can result from many different mechanisms other than lossof the receptor or target molecule of the antibacterial agent. Asnon-limiting examples, an hmw bacteriocin of the disclosure would not besubject to a bacterial efflux pump to remove the bacteriocin from thecellular environment and would not be subject to a bacterial nucleicacid deactivation mechanism.

Having now generally described the inventive subject matter, the samewill be more readily understood through reference to the followingexamples which are provided by way of illustration, and are not intendedto be limiting of the disclosure, unless specified.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed subject matter.

Example 1 Modified hmw Bacteriocins Containing a Fusion Protein a)Complementation System

To facilitate the preparation of a modified hmw bacteriocin as describedherein, construction of a system to complement tail fibers in trans wasestablished. Using the R2 pyocin as a representative model, creation ofa deletion of the R2 prf15 coding sequence in the P. aeruginosa PAO1genome was used to create a platform in which a complementing tail fiberprotein, such as a modified prf15 gene product, was expressed in trans.

Generally, the deletion was made by the method of Hoang et al. to createP. aeruginosa strain PAO1Δprf15. The prf16 coding region, SEQ ID NO:4,for the R2 chaperone overlaps the end of the R2 prf15 gene by 8nucleotides and the ribosome binding site lies within the prf15 codingregion, SEQ ID NO:3. The PRF16 protein, which is not necessarilyincorporated into the pyocin structure, has been reported to be requiredfor assembly of the trimeric tail fiber and thus for maximumbactericidal activity (FIG. 8 and Nakayama et al., 2000). Therefore,both the transcription start site for prf16 and its ribosome bindingsite were left intact such that the chaperone would be produced uponinduction of the modified pyocin construct encoding a “tail-less,”defective pyocin.

Briefly, an in-frame deletion of codons 11-301 of prf15 was made in PAO1as follows. A 1.1 kb KpnI-AgeI fragment upstream of the desired deletionwas amplified by PCR from PAO1 genomic DNA using primers AV085(5′-GCTTCAATGTGCAGCGTTTGC) (SEQ ID NO:46), and AV088(5′-GCCACACCGGTAGCGGAAAGGCCACCGTATTTCGGAGTAT) (SEQ ID NO:47), and a 2.2kb AgeI-EcoRI fragment was amplified using primers AV087(5′-ATACTCCGAAATACGGTGGCCTTTCCGCTACCGGTGTGGC) (SEQ ID NO:48) and AV086(5′-TCCTTGAATTCCGCTTGCTGCCGAAGTTCTT) (SEQ ID NO:49). The resultingrestriction fragments were cloned into the KpnI and EcoRI sites ofpEX18Gm (Hoang et al) to make pEXGm-Δprf15. The finished construct wastransformed into strain PAO1 by electroporation (Chuanchuen et al).Integrants were selected with 100 μg/ml gentamicin, and segregants werethen selected in media containing 5 μg/ml sucrose and lacking NaCl andgentamicin. Deletion candidates were confirmed by PCR analysis, pyocininduction, and sequencing of a PCR-amplified fragment.

Strain PAO1Δprf15 grows similarly to its parent strain, PAO1, and thepyocin encoding genes remain inducible through the SOS response, leadingto lysis of the cell. While there appears to be some production ofpyocin gene products, stable “tail-less” pyocin particles were notproduced from PAO1Δprf15.

R2 pyocin prf15 was expressed in trans by first cloning the codingsequence into the broad host range Pseudomonas/E. coli shuttle vector,pUCP30T. See FIG. 9. In some initial constructs, transcription wasdriven constitutively or under lacI control from the tac promoter. Butin other constructs, transcription was modified to be regulated with anendogenous prf15 promoter such that expression would be regulatedthrough the SOS response. This permitted the expression of the modifiedprf15 gene to be induced synchronously with the expression of the otherpyocin genes residing in the PAO1Δprf15 genome.

Briefly, the broad host-range vector pUCP30T (Schweizer, H. P et al) wasmodified by filling in the unique BspHI site to form pUCP30TΔBsp. A tacpromoter was amplified by PCR from an MTD expression vector (a gift fromJeffery F. Miller, UCLA) using primers AV110(5′-TTTATTAGCGGAAGAGCCGACTGCACGGTGCACCAATG) (SEQ ID NO:50) and AV114(5′-CCCTCGAATTCATGAATACTGTTTCCTGTGTGAAATTG) (SEQ ID NO:51), then clonedinto pUCP30TΔBsp to create pUCP-tac.

The R2 PRF15 coding region was amplified from a subclone using primersAV118 (5′-CTTCCTTTCATGACGACCAATACTCCGAA) (SEQ ID NO:52) and AV116(5′-ACCACGAATTCTTCATCGTCCAAATGCCTC) (SEQ ID NO:53), while R2 prf15 andprf16 were amplified using primers AV118 and AV086(5′-TCCTTGAATTCCGCTTGCTGCCGAAGTTCTT) (SEQ ID NO:49). The amplifiedfragments of prf15 and prf15/16 were cloned into pUCPtac digested withBspHI and EcoRI to yield pUCP-tac-prf15 and PUCP-tac-prf15/16.

For expression using the endogenous prf15 promoter, prf15 and prf16 wereamplified together with the 1088 bp sequence upstream of prf15 from asubclone using primers AV107 (5′-CACCATCTAGACAATACGAGAGCGACAAGTC) (SEQID NO:54) and AV091 (5′-TCCTCAAGCTTACGTTGGTTACCGTAACGCCGTG) (SEQ IDNO:55) and cloned into pUCP30T digested with XbaI and HindIII to createpUCP-R2p-prf15/16.

Bacteria in log phase suspension growth and containing the expressionplasmids were treated with 3 μg mitomycin C/ml to induce pyocinproduction. Stable pyocins were produced upon induction with yieldssimilar to that of wild type PAO1. The pyocins had the same bactericidalspectrum and level of activity as R2 pyocin produced from PAO1. Thus,production of a stable pyocin complex required the expression of a tailfiber protein in addition to expression of the other pyocin encodinggenes, and expression of the tail fiber gene in trans was sufficient.

When prf15 was expressed constitutively from the tac promoter, cellgrowth was markedly slower than when it was regulated by lacI or theendogenous promoter. Although it appears that production of PRF15 alonein the cell is detrimental, yields of pyocins generated from bothpromoters are comparable.

A plasmid construct was prepared from which R2 prf16 was co-expressedwith R2 prf15 to insure proper temporal expression prf16 for folding ofPRF15 expressed in trans.

b) Recombinant hmw Bacteriocins

As described herein, five different R-type pyocins, based on spectra andtermed R1-5, have been recognized. Because gene sequences encoding thetail fiber proteins were known only for R1 (SEQ ID NO:1) and R2 (SEQ IDNO:3), PCR was used to isolate and sequence the R3 (SEQ ID NO:5), R4(SEQ ID NO:7), and R5 (SEQ ID NO:9) pyocin tail fiber genes along withtheir cognate chaperone encoding sequences present in their producerstrains, SEQ ID NO:6, 8, and 10, respectively. The chaperone genes ofpyocins R1 and R2 were also cloned and sequenced, SEQ ID NO:2 and 4,respectively. To confirm the hypothesis that the tail fiber dictatesspectra, the sequences encoding R1, R3, R4, and R5 pyocin tail fiberproteins were obtained and expressed in trans in PAO1Δprf15 such thatthey would be incorporated into the R2 pyocin structure. Each of theresulting recombinant strains was then induced to produce pyocins andthe spectrum of each was determined by spot assays, as shown in FIGS. 2and 8.

c) Fusion Proteins as Functional Tail Fibers on Pyocins R2-P2

A fusion of the R2 tail fiber prf15 gene and bacteriophage P2 gene Hsequences was created, expressed and used to produce additional modifiedhmw bacteriocins of the disclosure. Bacteriophage P2, which infects manyE. coli strains, has a tail fiber encoding gene H, (SEQ ID NO:25) thathas significant sequence similarity to R2 prf15 (SEQ ID NO:3),particularly at the N-terminus-encoding portion. The portion of gene Hencoding the C-terminal 551 amino acid residues of the P2 tail fiberprotein, which is the putative region conferring target specificity(RBD), was fused to the portion of prf15 encoding the 164 amino acidN-terminal baseplate-binding (BPAR) portion of R2 PRF15 to encode amodified tail fiber protein (SEQ ID NO:27).

Bacteriophage P2 also encodes a putative tail fiber chaperone, encodedby gene G (SEQ ID NO:26), similar to that encoded by R2 pyocin prf16(SEQ ID NO:4), and the chaperones of many of the other myoviridaephages. Because it is likely that the gene G encoded chaperone isimportant for folding the C-terminal portion of the P2 tail fiberprotein in the fusion, constructs were made to co-express P2 gene G.

The portion of R2 prf15 encoding amino acids 1-164 was amplified from asubclone using primers AV118 and AV127(5′-TTCTTTAAGCTTTTCCTTCACCCAGTCCTG) (SEQ ID NO:56) and was digested withBspHI and HindIII. The portion of P2 gene H encoding amino acids fromposition 158-669 was amplified from a P2 phage stock (Richard Calendar)using primers AV124 (5′-CCTCCTGAATTCTTATTGCGGCATTTCCG) (SEQ ID NO:57)and AV126 (5′-TCCTTCGAATTCTTACACCTGCGCAACGT) (SEQ ID NO:58). P2 gene H158-669 plus gene G was amplified using primers AV124 and AV125(5′-CCTCCTGAATTCTTATTGCGGCATTTCCG) (SEQ ID NO: 59). Each of the PCRproducts from P2 were digested with HindIII and EcoRI. pUCP-tac-R2-P2Hwas created by cloning the prf15 fragment encoding the 1-164 amino acidfragment together with the P2 gene H fragment encoding the 158-669 aminoacid fragment into pUCP-tac digested with BspHI and EcoRI.pUCP-tac-R2-P2HG was generated by cloning the prf15 fragment encodingthe 1-164 amino acid fragment together with the P2 gene H fragmentencoding the 158-669 amino acid fragment plus gene G into pUCP-tacdigested with BspHI and EcoRI.

Briefly, PAO1Δprf15 was transformed with the prf15-P2 gene H fusionconstructs and pyocin production was induced with mitomycin C. Pyocinparticles were purified and tested for activity by spot tests and by thebacterial survival assay (see FIG. 2). The purified pyocin particlescontaining the R2-P2 fusion tail fiber had bactericidal activity againstE. coli strain Cla but were incapable of killing P. aeruginosa strain13s. Furthermore, the expression of P2 gene G was needed to produceactive pyocin. This supports the hypothesis that the chaperone isrequired for proper folding of the C-terminal portion of the tail fiber,as shown in FIG. 8.

The abilities of a range of different R2-P2 tail fiber protein fusionsto form functional pyocins that kill E. coli Cla were explored by aseries of different R2-P2 fusions. Representative examples of thesefusions are shown in FIGS. 4-7, along with the indication of theirresulting bactericidal activities against E. coli C1a.

d) Additional Fusion Proteins as Functional Tail Fibers on PyocinsR2-L-413c

An additional modified hmw bacteriocin has been produced to target Y.pestis. L-413c is a yersiniophage that infects most strains of Y. pestis(Richard Calendar, personal communication). Most of the L-413c genome ishighly similar to P2 with the notable exception of its tail fiber geneH, SEQ ID NO:28, which has diverged considerably from that of P2.Without being bound by theory, and offered to improve the understandingof the disclosure, variation in the tail fiber gene H, and thus theencoded protein, is the feature that most likely accounts for itsdiffering host range.

The N-terminus of L-413c gene H (SEQ ID NO:28), however, sharesconsiderably sequence similarity to its P2 counterpart (SEQ ID NO:25),likely due to its function of baseplate binding. A fusion wasconstructed to create a fusion tail fiber with the N-terminal 1-164amino acids from R2 PRF15 fused to the C terminal (positions 158-913)portion of the L-413c tail fiber to create a modified tail fiber, asshown in FIG. 10 (SEQ ID NO:30). The fusion was expressed in PAO1Δprf15along with the L-413c tail fiber cognate chaperone, gene G (SEQ IDNO:29), as described above. After induction, the produced pyocinparticles killed Y. pestis KIM as well as E. coli C and thus had akilling spectrum analogous to the host range of yersiniophage L-413c.The modified pyocins did not kill any of the tested Pseudomonas strains.

e) Additional Fusion Proteins as Tail Fibers on Pyocins R2-VHML

A further modified hmw pyocin particle has been made with a novel fusiontail fiber created between the P. aeruginosa R2 pyocin tail fiber BPAR(encoded by prf15) and one of the two tail fiber genes (SEQ ID NO:21 AND22) of Vibrio harveyi Myovirus-Like (VHML) bacteriophage. We fused thediversifiable portion of the tail fiber (Oakey and Owens, 2000; Oakey etal., 2002; Doulatov et al. 2004) to the R2 pyocin tail fiber. The orf35gene [SEQ ID NO:22] and that of its presumed cognate chaperone, orf38[SEQ ID NO:23], were synthesized based on new DNA sequence data obtainedfrom Dr. Oakey's VHML phage provided to the Australian National GenomeCenter in Brisbane. A series of fusions was generated between R2 prf15and the orf35 gene, and the fusions were co-expressed with orf38 inPA01Δprf15. For example, the fusion between BPAR R21-164 and VHML Orf3526-410 formed robust R-type pyocin particles that could be purified andrevealed the R-type pyocin proteins including fusion tail fibers of theexpected size when analyzed by SDS PAGE.

Data generated in our laboratory has shown that with one exception, theonly chimeric R-type pyocin tail fiber structures forming R-type pyocinparticles that can be purified and reveal appropriate proteins on SDSPAGE were those that exhibit bactericidal activity on the expectedtarget bacteria. The one exception has been this one chimeric R2prf15-VHML orf35 fusion. This chimeric R-type pyocin preparation was notbactericidal for any of the Vibrio species tested, but the strain of V.harveyi from which the lysogenic VHML bacteriophage was isolated for DNAsequencing cannot be imported to the U.S. because of its pathogenicityto crustacean and oysters. We conclude that we have generated an“orphan” chimeric R2 prf15-VHML orf35 pyocin and that the resultingmodified hmw bacteriocin with the VHML-derived RBD can be subjected todiversification by the natural DGR of VHML.

f) Additional Fusion Proteins as Functional Tail Fibers on PyocinsR2-V10

Bacteriophage phiV10 belongs to the podoviridae group and can infectmost common E. coli O157:H7 strains (Waddell and Poppe, 2000; GenbankNC_(—)007804). Phage phiV10 does not encode a tail fiber like that ofphage P2 or R-type pyocins but instead encodes a tail spike-like proteinsimilar to that of bacteriophages P22 and epsilon15. These tail spikesare globular proteins that typically are polysaccharide-specific anddegrade the surface polysaccharide structures to which they bind. It islikely that the phiV10 tail spike specifically recognizes, binds to anddegrades the O157 antigen on the surface of the pathogenic E. coliO157:H7.

We deployed as RBD various C-terminal portions, such as aa 204-875,211-875, and 217-875, of the phiV10 tail spike protein (SEQ ID NO.:60)by genetically fusing them individually to N-terminal BPAR encodingportions, such as aa 1-161 and 1-164, of the R2 pyocin tail fiber. Whenthree of these recombinant fusion genes (R2 Prf15 1-164:V10 tail spike204-875-[SEQ ID NO.: 67]; R2 Prf15 1-164:V10 tail spike 217-875 [SEQ IDNO.: 68]; and R2 Prf15 1-161:V10 tail spike 211-875 [SEQ ID NO.: 69])were expressed individually in the appropriate Pseudomonas strain(PAO1Δprf15), those pyocin particles produced and released hadincorporated into their structure functional R2-V10 tail fusions. Thesechimeric pyocin particles had bactericidal activity against all eight E.coli O157:H7 strains in our laboratory but did not kill any other E.coli strains, including mutants of EDL933 that have a defective 0157antigen. Furthermore, the chimeric R2-V10 pyocin digested the 0157antigen as evidenced by SDS PAGE of LPS extracted from E. coli EDL933.We have therefore created a recombinant R-type pyocin that specificallyrecognized and digested the 0157 antigen, a known virulence factor of E.coli O157:H7, and killed specifically E. coli O157:H7 strains.

To determine whether mutants resistant to R2-V10 pyocin that mightemerge from E. coli O157:H7 treated with R2-V10 pyocin would loose their0157 virulence factor, we selected, with and without prior chemicalmutagenesis, EDL933 E. coli mutants resistant to R2-V10 pyocins. The LPSstructures of seven independent mutants were analyzed by SDS PAGE todetermine whether they had altered O-antigen structures. Each of theseven had different qualitatively or profoundly quantitatively altered0157 antigens demonstrating that resistance to R2-V10 did result in lossof the virulence factor, 0157, from the E. coli O157:H7 pathogen.

When modeling the phiV10 tail spike protein with the Quickfire software(Imperial College, London), which utilizes a suite of protein structureanalysis programs, we found that it predicted in the C-terminal 422amino acids [SEQ ID NO: 60] structural homology with a galacturonase[SEQ ID NO: 66]. This explained the ability of portions of the V10 tailspike when fused to the N-terminal portion of pyocin Prf15 [such as SEQID NO:67, 68, 69] to enable the recombinant r-type pyocin to kill E.coli 0157 by binding to the 0157 antigen, which containedalpha-D-Gal2NAc, but not to kill the TEA026 mutant known to lackalpha-D-Gal2NAc in its O-antigen or the other 7 classes of EDL933mutants described above. Quickfire also showed that the phiV10 tailspike has structural homology to the endorhamnosidase of phage P22 tailspike [SEQ ID NO: 70], a phage tail protein known to bind and degradethe Salmonella O-antigen. Thus, the fusion of protein with such acatalytic function to the N-terminal BPAR of the tail fiber of an R-typepyocin conveyed to the recombinant R-type pyocin the ability to utilizethe enzyme's substrate binding property to target and kill bacteriaexpressing the enzyme's substrate on its surface.

Thus, R-type pyocins have been engineered to have differentspecificities using tail proteins from phages with tail structuresnaturally dissimilar to R-type pyocins, thus creating unnatural R-typepyocins.

g) Additional Fusion Proteins as Functional Tail Fibers on PyocinsR2-PS17

Pseudomonas aeruginosa phage PS17 was obtained from the Félix d'HérelleReference Center for Bacterial Viruses, Universtié Laval, Canada. PS17plaques were produced by infection of Pseudomonas aeruginosa strainPML14. PML14 cells lysogenic for phage PS17 were isolated by streakingcells from a plaque onto fresh tryptic soy agar plates. Lysogeny wasverified by colony PCR amplification with primers AV168 and AV167, whichwere designed from Genbank sequence BPSFIFII. An overnight culture ofthe lysogenic cells was diluted 1:100 in 200 ml tryptic soy broth andshaken (225 rpm) at 37° C. until the optical density at 600 nm wasapproximately 0.2. Mitomycin C was added to a final concentration ofμg/ml, and the culture was shaken a further 5 hours, at which time lysiswas apparent. Deoxyribonuclease (Invitrogen, 1 unit/40 ml) was added,and the lysate was incubated at 37° C. for 30 minutes to reduceviscosity. The debris was then removed by centrifugation at 12,000 rpmfor 30 minutes in a Beckman JA-25.50 rotor. The lysate was titered onstrain PML14 and determined to be approximately 8×10⁹ pfu/ml. Phage DNAwas isolated from 40 ml of the cleared lysate using a Qiagen Lambdaminiprep kit, following the manufacturer's instructions and using 3columns from the kit. The DNA was resuspended in a total of 40 μl.

Primer sequences (listed 5′ to 3′): AV168 (SEQ ID NO: 72)TCACGGTAACGAATGTGGACG AV167 (SEQ ID NO: 73) TTTCAGCCAGTTGGTCGACAC AV140(SEQ ID NO: 74) CCTGACGGATGGCCTTTTCTATTATCACTGCCCGCTTTCCAGTCG AV141 (SEQID NO: 75) TTTCTTTGCTCTTCCGCTAGAAGGCCATCCTGACGGATGGCCTTTTCT AV027 (SEQID NO: 76) TTTCTGCTCTTCAAGCCGACACCATCGAATGGTGCA AV169 (SEQ ID NO: 77)TTTATTAGCGGAAGAGCCACGCGTGACTGCACGGTGCACCAATG AV114 (SEQ ID NO: 78)CCCTCGAATTCATGAATACTGTTTCCTGTGTGAAATTG AV238 (SEQ ID NO: 79)AACCCACGAAGACCTCATGAGCACCAATCAATACG AV047 (SEQ ID NO: 80)CGCCAGGGTTTTCCCAGTCACGAC PRF13-F (SEQ ID NO: 81) TATCGAGAACTGCTGCTGCGGGAV086 (SEQ ID NO: 82) TCCTTGAATTCCGCTTGCTGCCGAAGTTCTT AV118 (SEQ ID NO:83) CTTCCTTTCATGACGACCAATACTCCGAA AV287 (SEQ ID NO: 84)TCGGTAATGCCGTACCCGCCCAGGGTGGTCGGATTGCTGC AV286 (SEQ ID NO: 85)GCAGCAATCCGACCACCCTGGGCGGGTACGGCATTACCGA AV293 (SEQ ID NO: 86)AAACCAAGAGCTCTTAGTTGGTGCCTTCTTCGGC

Based on the restriction map in Shinomiya and Ina (1989), we cloned andsequenced a ˜4.2 kb BglII fragment. A 1 μl sample of PS17 phage DNA wasdigested with BglII then electrophoresed in an agarose gel. Theappropriate fragment was excised from the gel, purified, and ligatedtogether with pUC19 vector DNA digested with BamHII. Competent E. colicells were transformed with the ligation products and transformants wereselected on LB agar plates containing 100 μg/ml carbenicillin. Plasmidminipreps were prepared and used for DNA sequence analysis. Twooverlapping open reading frames (SEQ. ID. NO.: 71) were found withsignificant homology to various tail fiber genes and chaperones. Inplasmid pUC19-PS-B3, there were approximately 53 bp between thetermination codon of the presumed chaperone open reading frame and theSacI restriction enzyme recognition site derived from the vectorpolylinker.

pUCP30T was digested with BspHI, the ends were made blunt by treatmentwith the Klenow fragment of DNA polymerase I in the presence of dNTPs,and the vector was religated to form pSW107, which lacked a BspHI site.

A lacI^(q) gene and a rrnBT2 terminator sequence were amplified by atwo-step PCR using a suitable plasmid vector containing lacI^(q), suchas pMAL-c2E, as template and primers AV140 and AV027 in the first stepand primers AV141 and AV27 in the second step. The PCR product wasdigested with SapI and ligated together with SapI-digested pSW107 toform plasmid pDG19.

A tac promoter was amplified by PCR using a suitable plasmid vectorpGEX-2T as template and primers AV169 and AV114. The PCR product wasdigested with SapI and EcoRI and ligated together with SapI- andEcoRI-digested pDG19 to form plasmid pDG35.

The presumed tail fiber and chaperone open reading frames were amplifiedby PCR from plasmid pUC19-PS-B3 using primers AV238 and AV047. The PCRproduct was digested with BspHI and SacI and ligated together withBspHI- and SacI-digested pDG35 to form pDG65.

A DNA fragment containing R2 prf15 and prf16 was amplified by PCR fromPAO1 genomic DNA using the primers PRF13-F and AV086. The PCR productwas cloned using a “Zero Blunt TOPO PCR Cloning Kit for Sequencing” fromInvitrogen. The resulting clone was designated pTOP0—R2.

A fragment of the R2 prf15 open reading frame representing codons 1-223was amplified by PCR using pTOP0—R2 as template and primers AV118 andAV287. A fragment of PS17 presumed tail fiber open reading framerepresenting the C-terminal portion from codon 220 through thetermination codon after codon 779 plus the presumed chaperone openreading frame was amplified by PCR using pUC19-PS-B3 as template andprimers AV286 and AV293. The resulting PCR products contained 20 bpoverlapping sequences. The two fragments were assembled by overlap PCRusing primers AV118 and AV293. The resulting fragment was digested withBspHI and SacI and ligated together with BspHI- and SacI-digested pDG35to form plasmid pSW122.

PS17 plaques normally have a turbid “bulls-eye” appearance on a lawn ofsensitive cells. PS17 phages were plated on strain PML14, and a few rareplaques with a clear appearance were picked. The clear-plaque phage werepurified by replating and picking isolated plaques with a clearappearance. An isolate designated PS17-c5 was chosen for further use.

P. aeruginosa strain PAO1 was deleted of nucleotides 10-2067 of itsprf15 coding sequence (SEQ. ID. NO.: 3) by a method analogous to thatdescribed to create PAO1Δprf15 in Example 1 to create PAO1-mΔprf15. A 50ml culture of PAO1-mΔprf15 in tryptic soy broth was shaken at 37° C.until the optical density at 600 nm was approximately 0.2. One plaque ofPS17-c5 was transferred from a plate to the liquid culture. After anadditional 3 hours shaking at 37° C. the optical density at 600 nmdropped, and lysis was apparent. The culture was then left to shakeovernight (16 hours). Following the overnight incubation, the culturehad become turbid again. A sample of the culture was inoculated onto atryptic soy agar plate and incubated overnight. Colonies were checkedfor PS17 lysogeny by colony PCR with primers AV168 and AV167 andappeared negative. A clonal isolate designated PAO1-mΔprf15-c5^(R) waschosen for further use as a host production bacterium for R2—PS17modified pyocins. Cells were made electrocompetent by a method similarto that described by Choi and Schweizer (2005) and transformed withpDG65 and pSW122. Transformants were selected and maintained with 100μg/ml gentamicin.

For expression of R2—PS17 pyocins, overnight cultures ofPAO1-mΔprf15-c5^(R) in tryptic soy broth supplemented with 100 μg/mlgentamicin were diluted 1:100 into G medium (Shinomiya, 1972) containing50 μg/ml gentamicin. The cultures were incubated at 37° C. with shaking(225 rpm) until the optical density at 600 nm was approximately 0.2.Mitomycin C was then added to a final concentration of 3 μg/ml, and thecultures were shaken at 37° C. 3-4 hours. Optionally, deoxyribonucleasewas added at 1 unit per 40 ml, and the lysate was incubated at 37° C.for 15-30 minutes to decrease viscosity. Debris was removed bycentrifugation at 12,000 rpm in a Beckman JA-25.50 rotor for 30 minutesat 4° C. The supernatant was transferred to a fresh centrifuge tube, andpyocins were pelleted at 22,000 rpm (approximately 58,500×g) for 1 hourat 4° C. The pellets were resuspended at 3% of the original volume in 10mM Tris-HCl pH 7.5, 50 mM NaCl and stored at 4° C.

The bactericidal activity of the recombinant “R2—PS17” pyocinpreparations were demonstrated by spotting dilutions on PS17-sensitivePseudomonas aeruginosa strains such as PML14.

h) Additional Fusion Proteins as Functional Tail Fibers on PyocinsR2-MTD

The major tropism determinant (MTD) of the Bordetella bacteriophageBPP-1 has a C-type lectin (CTL) domain, which serves as a bindingdeterminant for many different types of molecules and in many differentbiological contexts (Drickamer, 1999; McMahon et al., 2005). In BPP-1,MTD is incorporated as a homotrimeric globular domain located at the endof the phage tail, where it can bind to the surface protein pertactin, avirulence factor expressed on the outer surface of Bordetellabronchiseptica and Bordetella pertussis (Liu et al., 2004). In thiscontext, MTD is also the target of phage-mediated homing mutagenesis,which can result in the bacteriophage acquiring a novel bindingdeterminant for infecting its ever changing host.

Recent structural studies on the MTD domain and several of itsdiversified variants, have shown how the trimeric fiber tip forms arigid scaffold that can contain more than 10 trillion variant bindingligands (McMahon et al. 2005). Fusing the MTD domain onto the pyocintail fiber protein and then diversifying the MTD domain using the DGRsystem described by Miller and colleagues (Liu et al., 2004; Doulatov etal., 2004), creates a very large library of variants, from which toselect and obtain the genes encoding pyocin tails with altered bindingspecificity.

Example 2 Assays of Fusion Proteins a) Pyocin Purification and Assays

PAO1 or appropriate derivatives were grown shaking at 200 rpm at 37° C.in G medium supplemented with 50 μg/ml gentamicin when needed tomaintain plasmids. When the cultures reached OD600 of about 0.250,mitomycin C was added to a final concentration of 3 μg/ml. Cultures wereincubated for an addition 2.5 hours until lysis occurred. Five units (1unit/μl) of DNaseI (Invitrogen) was added to 200 ml of culture, and theculture was allowed to incubate an additional 30 mins. Debris wasremoved by centrifugation at 12,000 rpm in a Beckman JLA-16.250 rotorfor 1 hour. Saturated ammonium sulfate was slowly added, at a rate of 1ml/min, to the supernatant stirring on ice, to a final added volume of65 ml per 100 ml of the supernatant of the lysate. This was stored at 4°C. overnight. The ammonium sulfate precipitate was collected bycentrifugation at 12,000 rpm in a Beckman JA-25.50 rotor for 1 hour, 4°C., and the pellet was resuspended in 10 ml of TN50 buffer (10 mM tris,50 mM NaCl, pH 7.5). Pyocin particles in the resuspended solution werethen sedimented at 22,000 rpm (58,500×g) in a Beckman JA-25.50 rotor for1 hour, 4° C., and resuspended in 3-5 ml of TN50 buffer. Pyocin prepswere judged to be >90% pure by SDS polyacrylamide gel electrophoreticanalysis.

Quantitative pyocin assays were performed by counting bacterial survivalin a slightly modified method as described by Kagayama et al., 1964.Pyocin samples were incubated with target bacteria (approximately 1×10⁹CFU/ml) for 40 minutes at 37° C. The samples were then diluted andplated to count survivors. The number of pyocin particles is related tothe fraction of bacterial survivors in a Poisson distribution, m=−1nS,where m=the average number of lethal events/cell and S is the fractionof survivors. The total number of active pyocin particles/ml=m×cells/ml.Strain13s was the Pseudomonas aeruginosa used in these assays and is aclinical isolate resistant to many anitibiotics, but sensitive to all 5R-type pyocins. The E. coli target was C1a, kindly provided by RichardCalendar.

Semi-quantitative assays were also performed by a spot method wherepyocin samples were serially diluted in TN50 buffer and spotted on lawnsof target bacteria. After overnight incubation at 37° C., pyocinactivity could be observed by a clear zone of killing on the lawn. FIG.2 shows representative results from this assay format.

Example 3 Recombinant Bacteriophages to Screen Engineered Tail Fibers

The P4 bacteriophage was used as a surrogate to harbor a tail fiberfusion gene such that the genotype was coupled to the binding phenotypeof the tail fiber. This has allowed efficient selection, transductionand isolation of the gene for the desired tail fiber.

Bacteriophage P2 is a temperate coliphage which can infect other entericspecies, and can replicate in, but not infect, P. aeruginosa (Bertani &Six, 1988; Kahn et al., 1991). R-type pyocins are closely relatedgenetically and structurally to P2, and the P2 tail fiber protein,encoded by gene H, shows homology to PRF15 at the N-terminal portion,where base plate attachment occurs (Haggard-Ljungquist et al., 1992;Nakayama et al., 2000). Deploying the P2 or P4 bacteriophage as asurrogate phage, in which plasmid-encoded tail fibers were incorporatedin the phage particle in place of the P2 phage-encoded fibers, permittedthe display and selection of fusion fibers in a context that closelyresembled its intended functional context in the pyocin.

The tail fiber genotype was physically coupled to the binding phenotypein a transducing phage particle for genetic selection, similar to phagedisplay technology. When a P2 phage with an amber mutation in its fiberprotein gene H (made in an amber suppressor+E. coli) infected E. coliharboring a P4-based plasmid with a cos packaging site, which normallyacts as a signal for packaging bacteriophage genomic DNA (Ziermann &Calendar, 1991; Kahn et al., 1991), it packaged the cos-containing P4plasmid in the heads of newly synthesized P2/P4 phage particles. TheP4-based plasmid, FIG. 12, encoded and expressed the tail fusion gene.The fusion tail fibers expressed from the P4 plasmid in the P2 infectedE. coli were incorporated into the P2/P4 particles in place of thedefective (amber truncated) gene H product (P2 tail fiber protein). Uponlysis of the infected bacteria by the expression of the P2 holin andlysozyme, the released P2/P4-based transducing particles carried thecos-containing P4 plasmid encoding the tail fiber fusion gene ratherthan the P2 genome and had attached the recombinant fusion tail fibersrather than the amber truncated P2 tail fibers.

Specifically, plasmid pSW166 was constructed by replacing the regioncorresponding to bases 226-2594 of bacteriophage P4sid₁ (Shore et al.1977) with the 763 bp fragment consisting of the promoter and codingregion of aacC1 (gentamicin acetyltransferase 3-1) from plasmid pUCP30T(Schweizer, 2001; NCBI accession U33752) flanked by restriction sitesintroduced by PCR amplification (MfeI and KpnI next to the promoter andEcoRI next to the termination codon), cloned in the same orientation asthe P4 int gene.

Plasmid pDG211 was constructed by inserting between the MfeI and KpnIsites a 274 bp fragment derived by PCR amplification and consisting of aP4 PLE promoter (Deho et al., 1988) corresponding to bases 8585-8835(complementary strand) such that the promoter was in the same polarityas aacCI and NheI and NcoI sites were created between the KpnI site andthe PLE promoter.

DNA fragments derived by PCR amplification encoding amino acids 1-157 ofthe P2 gene H tail fiber gene and amino acids 218-875 of the phiV10 tailspike gene were inserted between the NcoI and KpnI sites of pDG211 tocreate pDG218, FIG. 12.

A 1 ml culture of E. coli C1a harboring plasmid pDG218 was grown toOD600 of 1.0, supplemented with 1 mM CaCl₂ and infected withP2amH72vir20 (Sunshine et al., 1971) at a multiplicity of infection ofapproximately 2. After a 10 minute pre-adsorption, the cells were shakenat 225 rpm at 37° C. for 50 minutes. The bacteria and debris wereremoved by centrifugation in a microcentrifuge for 1 minute, and thelysate supernatant was saved.

Cultures (200 μl) of each E. coli TEA026 and E. coli EDL933 (Ho andWaldor, 2007) target cells were supplemented with 2.5 mM CaCl₂ and 2.5mM MgCl₂. Supernatant (50 μl) from the lysate (previous step) was addedand preadsorbed for 10 minutes. The cells were then diluted with 700 μlbroth and shaken at 225 rpm at 37° C. for 1 hour. Aliquots (10 μl) ofeach cell suspension was plated on LB agar plates containing 15 μggentamicin/ml. The plates were then incubated overnight at 37° C. Whilean estimated 1000 colonies grew on the EDL933 plate, none grew on theTEA026 plate.

A control P4-based, negative control plasmid, pDG212, which wasconstructed to contain the complete, unfused P2 gene H tail fiber generather than the P2-V10 fusion gene as in pDG218, was similarly packagedfrom E. coli C1a after infection with P2amH72vir20. When 10 μl of thecontrol lysate was incubated with E. coli C1a on gentamicin-containingplates, 10,000 colonies grew but none appeared when the same controllysate was incubated with EDL933 on gentamicin containing plates. Thus,the false positive frequency for generating gentamicin-resistantcolonies of EDL933 from transfection with P4 particles that do notharbor the 0157-specific binding property is less than 10⁻⁴.

Transducing phage particles with the ability to bind cells and triggerthe bacteriophage injection mechanism confered gentamicin resistance tosuccessfully targeted bacteria, from which the selected fiber fusiongene was isolated from the plasmid after replication of the bacteriaunder gentamicin selection. The functional V10-based RBD gene isolatedby PCR was fused to the BPAR (aa 1-164) of R2 prf15, expressed in transin PA01Δprf15 and recombinant pyocins isolated and assayed forbactericidal activity on E. coli TEA026 and E. coli EDL933. As describedfor the P2/P4 particle, the RBD from V10 tail spike protein when fusedto BPAR from R2 pyocin PRF15, conveyed specificity to the resultingmodified pyocin such that it was bactericidal for EDL933 but not for themutamt TEA026, lacking the 0157 antigen. The tail fiber gene on the P4plasmid is easily further manipulated to create many fusion junctionsand to diversify the RBD in order to redesign and optimize the functionof the modified tail fiber RBD.

A related P4 virion particle was made to carry recombinant tail fibersgenerated by fusing an BPAR portion of P2 gene H encoding aa 1-158 tothe portion of the tail fiber gene of Pseudomonas phage, PS17, encodingan RBD of aa 164-779. The gene H—PS17 tail fiber fusion gene wasco-expressed with the latter's cognate chaperone from a P4-basedplasmid, pDG224, similar to pDG218. Once harvested from the lysate of aP2amH72vir20 infected E. coli C1a harboring pDG224, the P4 particlesconveyed by transformation gentamicin resistance to P. aeruginosa strainPML14 but not to P. aeruginosa strain PA14. PS17 phage also infected theformer but not the latter P. aeruginosa strain. Thus, the P4-basedsurrogate system has provided selection methods to couple recombinantgenotype to recombinant tail fiber binding phenotype even across generaof bacteria.

This approach also overcomes many of the difficulties imposed byC-terminal display of a trimeric protein when using conventional phagedisplay systems (Held & Sidhu, 2004). Bacteriophage P2 has tail fibersthat genetically and morphologically resemble those of pyocins (Nakayamaet al., 2000). Tail fibers attach to the base plates of P2 and pyocinsvia their N-termini, and there is significant sequence similarity of theN-termini of P2 and R2 pyocin tail fibers (Nakayama et al, 2000;Haggard-Ljungquist et al., 1992). Furthermore, the tail fiber gene ofthe P2-related phage, PS17, can complement the R2 pyocin tail fibergene, prf15 (Shinomiya, 1984; Shinomiya & Ina, 1989).

Alternatively, portions of the tail fiber gene orf35 of VHML phage ofVibrio harveyii (which like BPP-1 also contains a functioning DGR) isfused to the N-terminal domain of P2 gene H. This recombinantconstruction will then allow the P4-based selection of RBDs ofparticular interest, as described above, after the DGR-drivendiversification of the VHML VR embedded in the orf35 RBD.

Example 4 Methods to Recover the Desired Tail Fiber Gene

A P2 or P4 bacteriophage carrying an engineered tail fiber gene acted asa surrogate to couple pyocin tail fiber genotype to binding phenotype.By selecting or screening for specific binding phenotypes from thediversified or mutagenized libraries of the tail fiber genes harbored insuch surrogate bacteriophages, one can isolate the tail fiber genes thatencode a desired binding specificity. The selection may be carried outby single or multiple enrichment cycles of adsorbing the surrogatebacteriophages or transducing particles onto solid-phase targetmolecules, either by first removing undesired binders and thenisolating, from among the remaining surrogates, those that bind to theintended target molecules, or visa versa. Alternatively, the selectionmay occur by applying either binding step alone. Ultimately, thesurrogate exhibiting the desired binding phenotype can be subject to DNAextraction and isolation of the harbored tail fiber gene by cloningrestriction enzyme fragments or by PCR reactions using oligonucleotideprimers that bind specific DNA sequences peripheral to the diversifiedportion of the tail fiber gene.

Even though the surrogate phages or transducing P4 particles will notform plaques on the target-expressing bacteria, the infected ortransduced bacteria will still acquire antibiotic resistance, such as P4plasmid-encoded gentamicin resistance, along with the harbored plasmidor phasmid and therefore can be selectively grown and subsequentlyextracted to isolate the multi-copy plasmid and its desired tail fibergene.

These techniques permitted the identification and isolation of surrogatebacteriophages or transducing particles exhibiting the desired, specificbinding phenotypes from which we extracted and isolated the desired,specific, unnatural hmw bacteriocin tail fiber genes. Furthermore, thebinding of surrogates to mammalian molecules, cells or tissues can bedeployed to deplete from the libraries any genes encoding tail fibersthat might cause adverse events if incorporated into therapeutic hmwbacteriocins.

There is an available library of insertional mutant Pseudomonasaeruginosa bacterial strains differing from highly pathogenic parentalPA14 Pseudomonas aeruginosa only by the lack of expression of a seriesof specific virulence factors, one missing from each non-redundant,isogenic mutant (see the website atausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi). These isogenic mutantstrains provide tools for ensuring the specificity of the surrogatebacteriophages for the targeted virulence factors and not for otherprevalent surface molecules. For example, the population of surrogate P4bacteriophages can be incubated with a high density culture of aPseudomonas aeruginosa mutant missing a particular targeted virulencefactor in order to adsorb and deplete from a population of surrogatebacteriophages or transducing particles, those that bind to surfacemolecules present on both the isogenic mutant and the virulent parentalstrain. The depleted population will be enriched in surrogates bindingto the desired virulence factor. Once surrogate bacteriophages that dobind to and infect the bacteria expressing the particular virulence orfitness factor are isolated, each can be screened directly for itsinability to infect the isogenic mutant strain lacking the targetedfactor. The selected plasmid can be repackaged in surrogate transducingparticles and recycled any number of times through theadsorption-depletion and infection process to further enrich andeventually purify the pUC-based plasmid encoding the desired tail fibersfor targeting the virulence or fitness factor.

A tail fiber gene, recombinant or natural, encoded in a recombinant P4genome can be subject to mutagenesis, particularly in the portion of theRBD domain that confers specificity, by any one of several methodsfamiliar to one ordinarily skilled in the art. The mutagenized P4genomic plasmid is transformed at low multiplicity into E. coli C, andthe gentamicin-resistant transformants are subsequently infected byphage P2 amber H (P2amH72vir20). As described above, a library of P4virion particles will be packaged and produced and will have tail fiberswith mutant RBD portions, the gene for which will be encodedspecifically within the packaged recombinant P4 genome. Some of thesemutations will encode binding capabilities specific for a given targetreceptor on a target bacteria. The P4 genome harboring the mutant RBDwith the desired, and even rare, binding specificity can be selected byinfecting the target bacterial strain with the virion library andisolating gentamicin-resistant bacterial colonies. The resistantbacteria will harbor P4 genomes that encode a mutant RBD portion thathas acquired specificity for the target bacterial strain.

An example of selecting a rare desired binding phenotype and therebygenotype from a large population of undesired P4 particles wasdemonstrated by mixing different proportions of lysates of P4 particlesfrom E. coli C1a harboring pDG212 (P2 tail fiber) and E. coli Claharboring pDG218 (P2-V10 recombinant tail fiber). When the mixturescontained 0%, 1%, 99%, or 100% of the P4 particles with the P2-V10 tailfibers or the converse number of P4 particles with P2 tail fibers, theappropriate numbers of EDL933 or E. coli C1a transformed colonies grewon gentamicin containing agar plates. That is the P4 particles harvestedfrom E. coli harboring pDG218 could only transfect and conveygentamicin-resistance to EDL933 bacteria; while those harvested from E.coli harboring pDG212 could only transfect and conveygentamicin-resistance to E. coli C1a bacteria. The frequency of falsepositive growth, that is formation of gentamicin-resistant colonies ofE. coli EDL933 after attempted transformation with P4 frompDG212-harboring E. coli, was less than 10⁻⁵. The converse was alsoobserved, that is formation of gentamicin-resistant colonies of E. coliC1a after attempted transformation with P4 from pDG218-harboring E.coli, was less than 10⁻⁵.

The DNA encoding mutant RBDs with desired binding phenotype can beisolated by the PCR method using primers within the gene H sequence 5′to the RBD and sequences 3′ to the RBD but immediately outside the RBDcoding region. The selected RBD DNA sequence will be fused with the BPARportion of an R-type pyocin prf15 gene, such as that portion encoding aa1-164, and expressed in trans in bacteria such as PA01Δprf15 or aproduction strain as described below to make recombinant R-type pyocinswith a novel, desired binding and bactericidal specificity.

Example 5 Methods for Producing Engineered hmw Bacteriocins

The modified tail fiber gene is recombined either (i) into a plasmidunder a regulated promoter for expression in production bacteria alsoharboring, for example on a bacterial artificial chromosome (BAC), theR-pyocin gene cluster (including the endolysin genes) from which theresident prtR, prtN, prf15 and holin (prf9 or PA0614) genes have beendeleted or otherwise disabled, or (ii) into the pyocin clustercontaining BAC vector itself, using a plasmid-mediated allelic exchangereaction.

a) Expression of R-Type Pyocins in E. coli

The R2 pyocin gene cluster was cloned in four different variations usingfive different cloned fragments derived from PCR products.

Fragment 1 was amplified by PCR from PAO1 genomic DNA using primersAV461 and PRF13R, then digested with restriction enzymes EcoRI andHindIII. The resulting fragment represented bases 4267-7856 of Genbanksequence AB030825. Primer AV461 added an EcoRI site. This fragmentlacked genes prt-R and prt-N.

Fragment 2 was amplified by PCR from PAO1 genomic DNA using primersAV529 and PRF13R, then digested with restriction enzymes EcoRI andHindIII. The resulting fragment represented bases 2975-7856 of Genbanksequence AB030825. Primer AV529 added an EcoRI site. This fragmentcontained genes prt-R and prt-N.

Fragment 3 was amplified from PAO1 genomic DNA using primers AV333 andAV334, then digested with HindIII and NheI. The resulting fragmentrepresented bases 7856-14280 of Genbank sequence AB030825. This fragmentcontains a full-length prf15 gene.

Fragment 4 was amplified from PAO1-rΔprf15 genomic DNA using primersAV333 and AV334, then digested with HindIII and NheI. The resultingfragment represented bases 7856-9155 and 10028-14280 of Genbank sequenceAB030825. This fragment contained a prf15 gene with an 873 bp internaldeletion.

Fragment 5 was amplified from PAO1 genomic DNA using primers AV407 andAV404, then digested with NheI and PacI. The resulting fragmentrepresented bases 14,280-19,860 of Genbank sequence AB030825. PrimerAV404 added a PacI site.

A fragment of pBR322 (comprising nucleotides 2334-4353 of Genbanksequence SYNPBR322) including the origin of replication and theβ-lactamase gene was amplified using primers AV337 and AV338. Theresulting fragment was digested with NotI, and a multiple cloning sitewas created by ligating the NotI-digested vector with kinased andannealed oligos AV339 and AV340. The resulting plasmid was designatedpDG121.

PCR primer sequences: (SEQ ID NO: 87) PRF13-R GCACCGTTACCCGATCCGCGA (SEQID NO: 88) av333 TCGAGACGATTTACCAAGAGCTG (SEQ ID NO: 89) av334TTCCACGACCAGTCCGGAAAATG (SEQ ID NO: 90) av337TTTATTTGCGGCCGCGACGAAAGGGCCTCGTGATAC (SEQ ID NO: 91) av338TTTATTTGCGGCCGCAAATACCGCATCAGGCGCTCTTC (SEQ ID NO: 92) av339GGCCGCTTATTAACAAGCTTCACACACGCTAGCCCACCACGC (SEQ ID NO: 93) av340GGCCGCGTGGTGGGCTAGCGTGTGTGAAGCTTGTTAATAAGC (SEQ ID NO: 94) av404CCCCCCCTTAATTAACTTGAGTCAGGATGGACATG (SEQ ID NO: 95) av407AAGGCATTCGAGACCGTCAAG (SEQ ID NO: 96) av461TTTCCTTGAATTCGCTCGGCAATCTACAGACCGATG (SEQ ID NO: 97) AV529TTTCCCTGAATTCATTACTTGCCCACGCAGAAGGCGCTTTC

The plasmid pDG173 contained fragments 1, 3 and 5, inserted respectivelybetween the EcoRI and PacI sites of pDG121.

The plasmid pDG174 contained fragments 2, 3 and 5, inserted respectivelybetween the EcoRI and PacI sites of pDG121.

The plasmid pDG175 contained fragments 1, 4 and 5, inserted respectivelybetween the EcoRI and PacI sites of pDG121.

The plasmid pDG176 contained fragments 2, 3 and 5, inserted respectivelybetween the EcoRI and PacI sites of pDG121.

Chemically competent cells of E. coli strain BL21 (non-XDE3 lysogen;Novagen Cat. No. 69449-3) were transformed with plasmids pDG173, pDG174,pDG175 or pDG176. The retention of the plasmids was selected andmaintained with 50 μg/ml carbenicillin.

For expression of pyocins, overnight cultures of strain BL21 in LB brothsupplemented with 50 μg/ml carbenicillin were diluted 1:100 into Gmedium (Shinomiya, 1972) containing 25 μg/ml carbenicillin. The cultureswere incubated at 37° C. with shaking (225 rpm) until the opticaldensity at 600 nm was approximately 0.2. Mitomycin C was then added to afinal concentration of 33.3 ng/ml, and the cultures were shaken at 37°C. overnight (15-22 hours). The cultures still appeared turbid. Cellsand debris were removed by centrifugation at 12,000 rpm (approximately17,400×g) in a Beckman JA-25.50 rotor for 30 minutes at 4° C. Thesupernatant was transferred to a fresh centrifuge tube, and pyocins werepelleted at 22,000 rpm (approximately 58,500×g) for 1 hour at 4° C. Thepellets were resuspended at 3% of the original volume in 10 mM Tris-HClpH 7.5, 50 mM NaCl and stored at 4° C. The bactericidal activity of eachpreparation was assayed on strain 13s of P. aeruginosa. 10 μl of eachsample was electrophoresed on a 4-20% polyacrylamide tris-glycine SDSgel (SDS-PAGE) alongside molecular weight standards. The preparationsfrom the E. coli transformants containing pDG173 and pDG174 exhibitedpotent bactericidal activities and clear R-type pyocin protein subunitson SDS-PAGE analyses. The preparations from the E. coli transformantscontaining pDG175 and pDG176 did not exhibit bactericidal activity anddid not demonstrate substantive R-type pyocin protein subunits onSDS-PAGE analyses, all as predicted.

b) Expression of R-Type Pyocins in Pseudomonas fluorescens

Kinased and annealed oligos AV530 and AV531 were ligated into EcoRI- andHindIII-digested broad-host range plasmid vector pUCP30T (GenbankXXU33752). The resulting plasmid was designated pDG171.

Primer sequences: AV530 (SEQ ID NO: 98)AGCTgcggccgcGAATTCacgcgtAAGCTTactagtGCTAGCTTAATTAA AV531 (SEQ ID NO: 99)aattTTAATTAAGCTAGCactagtAAGCTTacgcgtGAATTCgcggccgc

The ˜15.6 kb EcoRI-PacI fragment from pDG173 was ligated into EcoRI- andPacI-digested pDG171 to create pDG193.

The ˜16.9 kb EcoRI-PacI fragment from pDG174 was ligated into EcoRI- andPacI-digested pDG171 to create pDG194.

The ˜14.7 kb EcoRI-PacI fragment from pDG175 was ligated into EcoRI- andPacI-digested pDG171 to create pDG195.

The ˜16.0 kb EcoRI-PacI fragment from pDG176 was ligated into EcoRI- andPacI-digested pDG171 to create pDG196.

Pseudomonas fluorescens (ATCC Cat. No. 13525) were made electrocompetentby a method similar to that described by Choi and Schweizer (2005), andtransformed with pDG193, pDG194, pDG195 or pDG196. Transformants wereselected and maintained with 100 μg/ml gentamicin.

For expression of pyocins, overnight cultures in tryptic soy brothsupplemented with 100 μg/ml gentamicin were diluted 1:100 into G medium(Shinomiya, 1972) containing 50 μg/ml gentamicin. The cultures wereincubated at 37° C. with shaking (225 rpm) until the optical density at600 nm was approximately 0.2. Mitomycin C was then added to a finalconcentration of 3 μg/ml, and the cultures were shaken at 37° C. 3-4hours. Debris was removed by centrifugation at 12,000 rpm (approximately17,400×g) in a Beckman JA-25.50 rotor for 30 minutes at 4° C. Thesupernatant was transferred to a fresh centrifuge tube, and pyocins werepelleted at 22,000 rpm (approximately 58,500×g) for 1 hour at 4° C. Thepellets were resuspended at 3% of the original volume in 50 mM NaCl, 10mM Tris-HCl, pH 7.5 and stored at 4° C. The bactericidal activity ofeach preparation was assayed on strain 13s of P. aeruginosa. 10 μl ofeach sample was electrophoresed on a 4-20% polyacrylamide tris-glycineSDS gel (SDS-PAGE) alongside molecular weight standards. Thepreparations from the E. coli transformants containing pDG193 and pDG194exhibited potent bactericidal activities and clear R-type pyocin proteinsubunits on SDS-PAGE analyses. The preparations from the E. colitransformants containing pDG195 and pDG196 did not exhibit bactericidalactivity and did not demonstrate substantive R-type pyocin proteinsubunits on SDS-PAGE analyses, all as predicted.

Upon induction of the pyocin genes and the engineered tail fiber gene,such as by inducing prtN directly via an engineered regulatable promotersuch as lac or tac, the host cells synthesize pyocins until theirnutrients are depleted and they cease growing (Young, Ry, 2006). Theproducing bacteria do not lyse in the absence of chloroform because theholin gene inactivation prevents cytoplasmic endolysin access to thebacterial cell wall, as is necessary for cell lysis. The exhausted cellsare harvested by centrifugation or filtration and then frozen until onedesires to harvest the soluble pyocins that have filled the cellularcytoplasm. Upon thawing, the inner cellular membrane ruptures, releasingendolysin to lyse the bacteria and thereby release the harvest ofmodified pyocins. The disruption of the bacterial membranes can beaccelerated or completed if necessary by the addition of smallquantities of chloroform to the aqueous solvent in which the bacterialpaste is thawed.

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Zolfaghar et al. “Mutation of retS, encoding a putative hybridtwo-component regulatory protein in Pseudomonas aeruginosa, attenuatesmultiple virulence mechanisms.” Microbes Infect. Jul. 15, 2005 Epubahead of print

All references cited herein are hereby incorporated by reference intheir entireties, whether previously specifically incorporated or not.As used herein, the terms “a”, “an”, and “any” are each intended toinclude both the singular and plural forms.

Having now fully described the disclosed subject matter, it will beappreciated by those skilled in the art that the same can be performedwithin a wide range of equivalent parameters, concentrations, andconditions without departing from the spirit and scope of the disclosureand without undue experimentation. While this disclosure has beendescribed in connection with specific embodiments thereof, it will beunderstood that it is capable of further modifications. This applicationis intended to cover any variations, uses, or adaptations of the subjectmatter following, in general, the principles of the disclosure andincluding such departures from the disclosure as come within known orcustomary practice within the art to which the subject matter pertainsand as may be applied to the essential features hereinbefore set forth.

1. A tail protein comprising a base plate attachment region (BPAR) froman R-type high molecular weight (hmw) bacteriocin tail fiber protein,wherein the BPAR comprises amino acids 1-161 of the N-terminal portionof the R-type bacteriocin tail fiber protein; and a heterologousreceptor binding domain (RBD) from a bacteriophage tail protein.
 2. Theprotein of claim 1 wherein the R-type hmw bacteriocin is an R-typepyocin and the bacteriophage is a podoviridae.
 3. An R-type highmolecular weight (hmw) bacteriocin having bactericidal activitycomprising a tail protein comprising a base plate attachment region(BPAR) from an R-type hmw bacteriocin tail fiber protein, wherein theBPAR comprises amino acids 1-161 of the N-terminal portion of the R-typebacteriocin tail fiber protein; and a heterologous receptor bindingdomain (RBD) from a bacteriophage tail protein.
 4. The R-type hmwbacteriocin of claim 3 wherein the BPAR comprises all or part of theamino acid sequence of SEQ ID NO:3 and the RBD comprises all or part ofthe amino acid sequence of SEQ ID NOS:60, 61, 62, 63, 64, 65, or
 70. 5.A method of compromising the integrity of a cytoplasmic membrane of apathogenic bacterium, said method comprising contacting said pathogenicbacterium with the bacteriocin of claim 3 to bind a virulence factor ofthe bacterium compromising the integrity of the cytoplasmic membrane. 6.The method of claim 5 wherein the pathogenic bacterium is E. coli O157.7. The method of claim 6 wherein compromising the membrane results insterilizing the E. coli O157.
 8. The method of claim 6 whereinbacteriocin is detectably-labeled and E. coli O157 is detected.
 9. Thebacteriocin of claim 3 wherein the expressed RBD binds a correspondingreceptor on a surface of a bacterial cell which leads to compromisingthe integrity of a cytoplasmic membrane of said cell.
 10. Thebacteriocin of claim 3 wherein the expressed RBD binds a virulence orfitness factor on a surface of a bacterial cell.
 11. The bacteriocin ofclaim 3 wherein said BPAR comprises amino acids 1-161, 1-164, 1-169,1-172, or 1-240 from SEQ ID NO:3.
 12. The bacteriocin of claim 3 whereinsaid RBD is from the C-terminus of a P2, L-413c, PS 17, BPP-1, CTX,VHML, CUS3, epsilon 15 HK620, sf6, ST64T, or phiV10 phage.
 13. Thebacteriocin of claim 12 wherein the C-terminus of the RBD is from about347 to about 755 amino acids in length.
 14. The bacteriocin of claim 3wherein the R-type bacteriocin is an R2-type pyocin and the RBDcomprises the C-terminal portion of a phiV10 bacteriophage tail spikeprotein.
 15. The bacteriocin of claim 3 wherein the RBD comprises aminoacids 204-875, 211-875, or 217-875 from SEQ ID NO:60.
 16. Thebacteriocin of claim 9 wherein the bacterial cell is E. coli O157. 17.The bacteriocin of claim 12 wherein the RBD comprises amino acids158-669 or amino acids 322-669 from SEQ ID NO:25; or amino acids 158-913from SEQ ID NO:28.
 18. The protein of claim 1 wherein said BPARcomprises amino acids 1-161, 1-164, 1-169, 1-172, or 1-240 from SEQ IDNO:3.
 19. The protein of claim 1 wherein said RBD is from the C-terminusof a tail fiber protein of P2, L-413c, PS 17, BPP-1, CTX, VHML, CUS3,epsilon15, HK620, sf6, ST64T, or phiV10 phage.
 20. The protein of claim19 wherein the RBD comprises amino acids 204-875, 211-875 or 217-875from SEQ ID NO:60.
 21. The protein of claim 1 wherein the R-type hmwbacteriocin is an R2-type pyocin and the RBD comprises the C-terminalportion of a phiV10 bacteriophage tail spike protein.
 22. The protein ofclaim 21 wherein the BPAR comprises ammo acids 1-161, 1-164, 1-169,1-172, or 1-240 from SEQ ID NO: 3, and the RBD comprises amino acids204-875, 211-874 or 217-875 from SEQ ID NO:60.
 23. The protein of claim1 wherein the BPAR comprises 95% sequence identity to amino acids 1-161,1-164, 1-169, 1-172, or 1-240 from SEQ ID NO:3.